Progress in Retinal and Eye Research 51 (2016) 1e40

Contents lists available at ScienceDirect

Progress in Retinal and Eye Research

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

Review

Gliaeneuron interactions in the mammalian retina

Elena Vecino a, *, 1, 2, F.David Rodriguez b, 1, Noelia Ruzafa a, 1, 2, Xandra Pereiro a, 1, 2,
Sansar C. Sharma c, d, 1
a
Department of Cell Biology and Histology, University of the Basque Country UPV/EHU, Leioa 48940, Vizcaya, Spain
b
Department of Biochemistry and Molecular Biology, E-37007, University of Salamanca, Salamanca, Spain
c
Department of Ophthalmology, Cell Biology and Anatomy, New York Medical College, Valhalla, NY 10595, USA
d
IKERBASQUE, Basque Foundation for Science at Dept. Cell Biology and Histology, UPV/EHU, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The mammalian retina provides an excellent opportunity to study gliaeneuron interactions and the
Received 30 January 2015 interactions of glia with blood vessels. Three main types of glial cells are found in the mammalian retina
Received in revised form that serve to maintain retinal homeostasis: astrocytes, Müller cells and resident microglia. Müller cells,
18 May 2015 astrocytes and microglia not only provide structural support but they are also involved in metabolism,
Accepted 2 June 2015
the phagocytosis of neuronal debris, the release of certain transmitters and trophic factors and Kþ up-
Available online 23 June 2015
take. Astrocytes are mostly located in the nerve fibre layer and they accompany the blood vessels in the
inner nuclear layer. Indeed, like Müller cells, astrocytic processes cover the blood vessels forming the
Keywords: retinal blood barrier and they fulfil a significant role in ion homeostasis. Among other activities, microglia
Retina
can be stimulated to fulfil a macrophage function, as well as to interact with other glial cells and neurons
Neurons
Glial cells
by secreting growth factors. This review summarizes the main functional relationships between retinal

List of abbreviations: AAV, adeno-associated virus; Ach, acetylcholine; AGA, amadori-glycated albumin; AGE, advanced glycation end-products; AhR, aryl hydrocarbon
receptor; AKT, protein kinase B; AMD, age-related macular degeneration; AP-1, activating protein-1; APC, antigen-presenting cells; Apo, apolipoprotein; AQP, aquaporin;
ASCL1, achaete-scute family bHLH transcription factor 1; Atoh7, atonal homolog 7; ATP, adenosine triphosphate; BBB, bloodebrain barrier; Bcl-2, B-cell lymphoma 2; BDNF,
brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; BV, blood vessel; CaMKII, Ca2þ/calmodulin-dependent protein
kinase; Car2, carbonic anhydrase II; CCL2, monocyte Chemotactic Protein-1; CCR2, CeC chemokine receptor type 2; CD45, cluster of differentiation 45; Cdc14A, cell division
cycle 14A; Cdk10, cyclin-dependent kinase 10; Ch, choroid; ChAT, choline acetyltransferase; CHX10, Ceh-10 homeodomain-containing homolog; CNS, central nervous system;
CNTF, ciliary neurotrophic factor; CNV, choroidal neovascularisation; CRALBP, cellular retinaldehyde-binding protein C; CREB, cAMP response element-binding protein;
CSF1R, colony stimulating factor 1 receptor; CSPG, chondroitin sulphate proteoglycans; CTGF, connective tissue growth factor; CX3CL1, chemokine (C-X3-C Motif) Ligand 1;
CX3CR1, CX3C chemokine receptor 1; DAPI, 40 ,6-diamidino-2-phenylindole; DAPT, g-Secretase inhibitor; DHA, docosahexaenoic acid; DKK3, Dickkopf-related protein 3; DR,
diabetic retinopathy; EAAT, excitatory amino acid transporter; ECM, extracellular matrix; ED-1, monoclonal antibody against glycoprotein CD68; EGF, epidermal growth
factor; EMV, extracellular membrane microvesicles; ESMV, embryonic stem cells microvesicles; FG, fluorogold; FGF, fibroblast growth factor; FKN, fraktalkine; G6P, Glucose-
6-Phosphate; GABA, g-aminobutyric acid; gadd45b, growth Arrest And DNA-Damage-Inducible Beta; GCL, ganglion cell layer; GDNF, glia-derived neurotrophic factor; GFAP,
glial fibrillary acidic protein; GFP, green fluorescent protein; GK, Goto Kakizaki; GLAST, glutamate-aspartate transporter; GLT1, glial Glutamate Transporter 1; Gpc, glypican;
GS, glutamine synthetase; hMGSC, human Müller glia stem cells; HSPG, heparin sulphate proteoglycan; IBA1, ionized calcium-binding adapter molecule 1; IF, intermediate
filaments; IFN, interferon; IGF, insulin-like growth factor; IL, interleukin; ILM, inner limiting membrane; INL, inner nuclear layer; IOP, intraocular pressure; IP3R2, IP3 receptor
type 2; IPL, inner plexiform layer; JNK, C-Jun N-terminal kinase; Kir, inwardly rectifying potassium channels; LCA, Leber congenital amaurosis; LDL, low-density lipoproteins;
LGN, lateral geniculate nucleus; LIF, leukaemia inhibitory factor; MAPK, mitogen-activated protein kinases; Mash1, mammalian achaete-scute homolog-1; MCP1, monocyte
chemoattractant protein-1; Megf10, multiple epidermal growth factor-like domains protein 10; Mertk, tyrosine-protein kinase Mer; MHC, major histocompatibility complex;
MMP, matrix metalloproteinases; mTOR, mechanistic target of rapamycin; NAD, nicotine adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; NF,
neurofilaments; NFL, nerve fibre layer; NFk&Bgr, uclear factor kappa-light-chain-enhancer of activated B cells; NGF, nerve growth factor; NMDA, N-Methyl-D-aspartate; NO,
nitric oxide; NPD1, neuroprotectin D1; NPPB, 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid; NV, neovascularisation; OIR, oxygen-induced retinopathy; OLM, outer limiting
membrane; ON, optic nerve; ONL, outer nuclear layer; OPC, oligodendrocyte precursor cells; OPL, outer plexiform layer; OPN, osteopontin; OS, outer segment layer; P2Y,
metabotropic purinergic; Pax6, paired box 6; PCAG, primary closed-angle glaucoma; PCG, primary congenital glaucoma; PE, pigment epithelium; PEDF, pigment epithelium-
derived factor; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PKB, protein kinase B; PLC, phospholipase C; POAG, primary open angle glaucoma; PSD95, postsynaptic
density protein 95; rd10, retinal degeneration 10; RFP, red fluorescent protein; RGC, retinal ganglion cells; RGCL, retinal ganglion cells layer; RNA, ribonucleic acid; ROS,
reactive oxygen species; RP, retinitis pigmentosa; RPE, retinal pigment epithelium; RT-PCR, reverse transcription polymerase chain reaction; RVD, regulatory Volume
Decrease; SELDI-TOF, surface-enhanced laser desorption ionization-time of flight; SNAP25, synaptosomal-associated protein 25; SP-A, surfactant protein A; Spbc25, spindle
pole body component 25; STAT, signal transducers and activators of transcription transient; STZ, streptozotozin; TAK1, transforming growth factor b-activated kinase 1; TCA,
tricarboxylic acid cycle; TGF&Bgr, transforming growth factor beta; Thy-1, thymocyte antigen 1; TN-C, tenascin C; TNF, tumour necrosis factor; TRK&Bgr, tropomyosin re-
ceptor kinase B; uPA, urinary-type plasminogen activator; VEGF, vascular endothelial growth factor; Vim, vimentin; WNT, Wingless-Type MMTV integration site family.
* Corresponding author.
1
Each co-author contributed equally (20%) to this article.
2
www.ehu.es/GOBE.

http://dx.doi.org/10.1016/j.preteyeres.2015.06.003
1350-9462/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40

Astrocytes glial cells and neurons, presenting a general picture of the retina recently modified based on experi-
Müller glia mental observations. The preferential involvement of the distinct glia cells in terms of the activity in the
Microglia retina is discussed, for example, while Müller cells may serve as progenitors of retinal neurons, astrocytes
Macrophages
and microglia are responsible for synaptic pruning. Since different types of glia participate together in
Retinal ganglion cells
certain activities in the retina, it is imperative to explore the order of redundancy and to explore the
Photoreceptors
Glaucoma heterogeneity among these cells. Recent studies revealed the association of glia cell heterogeneity with
Retinitis pigmentosa specific functions. Finally, the neuroprotective effects of glia on photoreceptors and ganglion cells under
Neuroprotection normal and adverse conditions will also be explored.
Neurotrophins © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
Plasticity license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Integrins
Extracellular matrix

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Origin and morphology of the retinal glial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1. Müller cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1.1. Origin and morphology of Müller cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1.2. Müller cell markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2. Astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.2.1. Origin and morphology of astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.2.2. Astrocyte markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2.3. Astrocytes main functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.3. Microglial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.3.1. Origin, morphology and distribution of microglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.3.2. Plasticity and functional microglia phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3.3. Representative molecular traits of microglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3.4. Identification of microglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3. Common functions of different glial cells in the retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.1. Formation of boundaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.2. Glia as a structural support for neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.2.1. Viscoelastic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.2.2. Architecture-mechanical forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.3. Volume regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.4. Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.4.1. Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.4.2. Lipid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.5. Extracellular matrix generators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.5.1. Laminin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.5.2. Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.5.3. Vitronectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.5.4. Fibronectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.5.5. Tenascin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.5.6. Proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.5.7. Intermediate Filaments (IFs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.5.8. N-Cadherin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.5.9. Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.6. Regulation of neuronal activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.6.1. Gamma-Aminobutyric acid (GABA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.6.2. Glutamate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.6.3. ATP and adenosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.7. Synaptic pruning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.8. Immune responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.9. Gliosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.10. Neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.10.1. Neurotrophic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.10.2. Other trophic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.11. Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.12. Repair and regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4. Specific functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.1. Light guidance of Müller cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.2. Progenitor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
5. Heterogeneity of retinal glial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
5.1. Astrocyte heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
5.2. Müller cell heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
6. Glia and retinal pathologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
6.1. Diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
6.2. Glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40 3

6.3. Age-related macular degeneration (AMD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

6.4. Retinitis pigmentosa (RP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
7. Conclusions, remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

1. Introduction to restore homeostasis, when they malfunction, these cells become

what can be considered a primary pathogenic element. Little is
The concept of neuroglia was first proposed by Rudolf Virchow known about the regulation of gliaeglia and gliaeneuronal in-
in 1858, in reference to the cellular elements that make up the teractions within the retina. However, since the retina is a well
connective tissue of the brain “nervekitt”. The Müller cell was structured organ within the Central Nervous System (CNS), studies
described as a radial fibre in the retina by Heinrich Müller in 1851, of the different roles of glial cells in health and disease have allowed
later becoming known as Müller glial cells. In 1893, the term researchers to understand some of their interactions with neurons.
astrocyte first appeared, used by Michael von Lenhossek in refer- The interactions between glia and neurons contribute to retinal
ence to the stellate morphology (from the Greek for star, astro, and homeostasis, and while some such interactions have been defined,
cell, cyte) of the cells that Camillo Golgi had previously seen in the we are still far from fully understanding how homeostasis is
nervous system, describing them as the “glue” of the brain. These maintained in the retina. Thus, in this review we will establish
cells were soon subdivided into fibrous and protoplasmic astro- some of the common features of glial cells in the retina and we will
cytes by Kolliker and Andriezen, yet their true diversity was not describe their possible interactions with neurons.
fully appreciated until Ramon y Cajal's drawings revealed their
extraordinary pleomorphism. Based on his histological studies,
several roles for this diverse class of cells were postulated, 2. Origin and morphology of the retinal glial cells
including a role in the maintenance of brain architecture, homeo-
stasis and nutrition. In 1919, oligodendrocytes (from the Greek for 2.1. Müller cells
few, oligo, branches, dendro, and cell cyte) and microglia were
recognized as separated cell types and categorised as neuroglia by 2.1.1. Origin and morphology of Müller cells
Pio del Rio-Hortega, one of the first scientists to propose that The Müller cell is the predominant glial element in the retina,
microglia are of mesodermal origin, and that they can migrate and representing 90% of the retinal glia. These are radially oriented cells
act as phagocytes (Kettenmann and Verkhratsky, 2008). that traverse the retina from its inner (vitreal) border to the distal
The vertebrate retina contains four types of glia, each of which end of the outer nuclear layer (ONL: Fig. 2). Although Müller cells
exhibits distinct morphological, developmental and antigenic share a lineage with retinal neurons, their time of birth appears to
characteristics. Müller cells are the most predominant glial be different. Recent studies found the surprising origin of Müller
element, followed by astrocytes and microglia. The fourth glial cell cells in the neural crest cell (Liu et al., 2014a). Birth dating of retinal
type, the oligodendrocyte, is seen only occasionally in the retina, neurons and Müller cells was determined by 3H-thymidine-label-
associated with myelinated ganglion cell axons in a few species like ling (Sidman, 1961) and all retinal cell types are generated in an
the rabbit. In this review, we will focus on general aspects of the orderly fashion, the sequence of which is largely conserved among
mammalian retina and thus, we will not mention oligodendrocytes, vertebrates (Vecino and Acera, 2015). Accordingly, ganglion cells
even though their activity and interactions with other astrocytes in are the first cells to be born, followed closely by horizontal cells,
the optic nerve, and with retinal ganglion cell (RGC) axons, might cones and displaced amacrine cells. At later stages, amacrine cells,
affect retinal function in some species (Fig. 1). bipolar cells, rod cells and Müller cells are generated, and indeed,
Despite studying the glia for more than a century the full influ- the general consensus is that Müller cells are the last cells to
ence of glial cells on neuronal centres is still not fully clear. While become post-mitotic (Cepko, 1993).
Cajal and others postulated several roles for glial cells (including The idea of a functional unit in which local interactions occur
maintaining brain architecture, homoeostasis, nutrition and regu- between a group of retinal neurons and their supportive glial cell
lating neuronal communication), we now know that glia are involved gave rise to the proposal of co-operativity among cells. This idea
in many other roles within the nervous system, for example influ- was based on developmental studies, where Müller cells were
encing synaptic connectivity by controlling the genesis and mainte- thought to perform many of the metabolic, ionic and extracellular
nance of synapses, and by modifying synaptic strength and plasticity. buffering requirements of neurons, with which they share a com-
Although Müller, astrocytes and microglial cells each have a different mon progenitor (Turner and Cepko, 1987). Later, an interesting
developmental origin, they share many functions within the retina. concept arose suggesting that co-operativity exists among cells that
Some of these activities are performed simultaneously, while others arise from a common stem cell to form a columnar array
are carried out by one or other of the glial cell types. More recently (Reichenbach et al., 1993). The idea is that the retina is constituted
glial cells have been established as a source of progenitors in the by many functional units in which local interactions occur between
brain, as not only can radial glia act as pluripotent neural precursors the group of retinal neurons and their supportive Müller glial cell,
but also, some astrocytes can re-enter the cell cycle and produce all limiting the sphere of influence of the latter. Thus, each Müller cell
types of brain cells, from neurons to macroglial cells. may only have to meet the requirements of its immediate neigh-
It is known that glial cells have intrinsic signalling systems that bours for the extracellular environment to remain stable in the face
can spread in the form of Ca2þ waves and that are correlated with of intense neural activity.
glutamate release. Among other functions, this signalling plays an In retinal sections, the processes of Müller cells are seen to
important role in immunity, angiogenesis and neuroprotection. ensheath the cell bodies and processes of retinal neurons, espe-
However, while neuroglia responds to injury as part of the defence cially after retinal damage (Hernandez et al., 2010). Thus, it seems
likely that the intimate association between these two cell types
4 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40

Fig. 1. Schematic drawing of the cellular components of the retina: glia and neurons. The different cell types are situated in a standard large mammalian retina. Note the in-
teractions between the cells and blood vessels (BV). Amacrine cells (A), astrocytes in green (AS), bipolar cells (B), cones (C), ganglion cells (G), horizontal cells (H), Müller cells in blue
(M), microglia in red (Mi), rods (R), cones (C). Note the location of the different layers of the retina (from the most internal to the outer layers): optic nerve (ON), nerve fibre layer
(NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), outer segment layer (OS), pigment
epithelium (PE), choroid (Ch).

enables glial cells to promote the formation of synapses, and it
would help maintain neuronal function by providing the nerve
terminals with energy substrates and neurotransmitter precursors
(Pfrieger and Barres, 1996). Ensheathing by Müller cells is only
possible to observe when membrane markers, like low affinity re-
ceptor p75, are used to stain the Müller cells, as it is not evident
with cytoskeletal markers (Fig. 3). As there are no specific astrocyte
membrane markers, and GFAP (glial fibrillary acidic protein) and
p75 co-localize in both Müller cells and astrocytes, it is possible that
at least some of the ensheathing membranes around RGCs could
belong to astrocytes. Moreover, in some species like dogs, the RGC
axons are packaged and intercalated between the end feet of
groups of Müller cells on their way to the optic nerve, forming a
nest like structure. By contrast, in species like the pig or minipig,
the RGC axons are intercalated between the Müller cells and such
axonal packages are not found (Fig. 2).
Although Müller cells from different species vary considerably
in shape, they do have some fairly universal features. At the level of
the outer limiting membrane (OLM), two processes extend in
opposite directions and Müller cells extend apical microvilli into
the sub-retinal space between the inner segments of photoreceptor
cells. The length and number of microvilli may vary between spe-
cies, probably in an inverse relationship to the degree of retinal
vascularisation (Uga and Smelser, 1973), and in some species Müller
cells also possess a cilium (Ennis and Kunz, 1986). In the opposite
direction, the inner process approaches the vitreal surface of the
neuroretina, forming a so-called endfoot that abuts the basal lam-
ina between the vitreous body and the neuroretina (the “inner
limiting membrane”, ILM). In the nuclear layers, the lamellar pro-
cesses of Müller cells form structures that envelop the cell bodies of
neuronal cells (Dreher et al., 1992; Hama et al., 1978; Reichenbach
et al., 1988, 1989). In addition, Müller cells are involved in the
structural organization of the blooderetina barrier (Reichenbach
et al., 1995). Blood capillaries are ensheathed by Müller cell pro-
cesses that act as a communication system for metabolic exchange
between the vasculature and neurons, in much the same manner as
postulated for brain astrocytes (Fig. 2D). However, the close phys-
ical association between Müller cell processes and retinal vessels
still leaves open the possibility that the Müller cell per se is an
essential component of the blooderetina barrier, and as such, it
may be capable of influencing its own permeability (Sarthy and
Ripps, 2001).
In summary, the Müller cell population accumulates in a dense,
regular pattern, although each cells can be considered as the core of a
columnar retinal “microunit” of neurons (Reichenbach and Robinson,
1995). This “strategic position” enables Müller cells to constitute an
anatomical and functional link between retinal neurons and the
compartments with which these need to exchange molecules, i.e.:
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40 5

Fig. 2. Müller cells. A, C and E are from dog retinas, B and D are from pig retinas, and F is from rat. (A) Müller cells are labelled in the retinal pigment epithelium (RPE) with
antibodies against vimentin (red) and RPE65 (green), and the nuclei were stained with DAPI (blue). (B) Retina section stained with antibodies against glutamine synthetase (GS). (C)
High magnification of a section at the level of the retinal ganglion cell layer (RGCL), labelled with antibodies against vimentin (red), showing the Müller endfeet and RGC axons
labelled with antibodies against neurofilaments (NF) in green. (D) High magnification of the Müller endfeet stained with antibodies against GS surrounding a blood vessel (BV) at
the nerve fiber layer level. (E) Müller cells and RPE of a dog retina section stained with antibodies against CRALB. (F) Rat Müller cells in culture marked with green fluorescent
protein (GFP). Scale bar for all pictures ¼ 50 mm.

retinal blood vessels, the vitreous chamber and the sub-retinal space.
Accordingly, Müller glia fulfil many crucial roles, supporting neuronal
development, survival and information processing (Reichenbach and
Bringmann, 2010; Sarthy and Ripps, 2001).
2.1.2. Müller cell markers
Müller cells can be easily identified by their characteristic
morphology, containing a well-developed endoplasmic reticulum
with varying amounts of glycogen granules (Hogan et al., 1971). A
prominent number of mitochondria are found at the endfeet
(Fig. 3). Müller cells can be labelled in living retinal preparations by
loading for a few minutes with vital dyes, such as Mitotracker or-
ange or CellTracker green. Their nuclei can be stained with
ethidium bromide, diamidino yellow or chromomycin A3, which
selectively penetrate Müller cells and have a marked affinity for
nucleic acids (Jeon and Masland, 1993; Laties, 1983). In culture,
Müller cell nuclei can be differentiated, as they are the largest of the
different retinal cell types.
In terms of immunohistochemical markers, the expression of
various proteins is restricted to astrocytes and Müller cells, like
vimentin and glial fibrillary acidic protein (GFAP), or selectively to
Müller cells, such as the cellular retinaldehyde-binding protein
(CRALBP), the glutamate-aspartate transporter (GLAST), glutamine
synthetase (GS), the Kir4.1 subtype of inwardly rectifying potas-
sium channels, aquaporin-4 (AQ4), pyruvate carboxylase, a-crys-
tallin, the small GTP-binding protein RhoB, glutamate
carboxypeptidase II, carbonic anhydrase C and microtubule-
associated protein 4 (Berger et al., 1999; Bunt-Milam and Saari,
6 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40

Fig. 3. Interactions between Müller cells and Retinal Ganglion cells (RGCs). (A) Retina section where RGCs are labelled with antibodies against ßIII Tubulin (green) and Müller cells
are labelled with antibodies against GFAP (red). (B) Electron microscopy detail of mitochondria in the interior of the Müller cell endfeet. (C) Retina section where the RGC are
labelled with antibodies against BDNF (green) and Müller cell membranes with antibodies against p75 (red). Note that RGCs were enveloped by p75-labelled Müller processes. (D)
Electron microscopy detail of RGC (G) wrapped in Müller cell processes (M). Scale bar A), C), D) ¼ 10 mm, B) ¼ 1 mm.

1983; Linser and Moscona, 1981; Moscona et al., 1985; Parysek
et al., 1985; Santos-Bredariol et al., 2006).
2.2. Astrocytes
2.2.1. Origin and morphology of astrocytes
Named on the basis of their stellate shape, astrocytes are almost
exclusively confined to the innermost retinal layers. The astrocytes
in the retina do not originate from the retinal embryonic epithe-
lium and thus, it is generally believed that these astrocytes migrate
from the optic nerve and probably enter the retina along with the
blood vessels (Stone and Dreher, 1987; Turner and Cepko, 1987;
Watanabe and Raff, 1988). In the optic nerve, as in other white
matter tracts, most astrocytes and oligodendrocytes are derived
from local mitoses (Skoff, 1990). The ventricular cavity disappears
in the evagination that forms the retina and optic nerve, and it is
believed that the mitotic precursors of the oligodendrocytes and
astrocytes migrate into the optic nerve from the sub-ependymal
germinal layer of the brain. According to the extent of myelina-
tion, there would appear to be species differences in the migration
of oligodendrocyte precursors. Thus, myelination stops at the
lamina cribosa in the human and rat, while in rabbit and dog
myelination extends beyond the lamina cribosa to reach the optic
nerve head. In some fish, where the lamina cribosa is absent,
myelination extends into the retina within the optic fibre layer
(Wolburg, 1981).
The presence and distribution of retinal astrocytes is correlated
with the presence and distribution of retina blood vessels (Fig. 4).
Thus, while avascular retinas contain no astrocytes, they are
distributed diffusely in those that are diffusely vascularized and
restricted to the vascularised regions of other retinas. This striking
correlation between vascularisation and the presence of astrocytes
led to the proposal that retinal astrocytes are immigrants that enter
the retina with its vasculature. While astrocytes may reside in
almost all regions of the retina, regional differences in their density
arise. Astrocytes are absent in retinal regions with extremely thin
nerve fibre layers, such as areas near the ora serrata, and they are
absent from the fovea centralis and the peri-foveolar retina of
primates. Moreover, the periphery of the retina has a thin nerve
fibre layer and it contains only a few dispersed astrocytes, the
number of which increases as the nerve fibre layer thickens. As-
trocytes send processes to both blood vessels and axons. Two
groups of astrocytes can be distinguished in the pig and minipig
retinas, those that are directly connected with blood vessels and
those that seem to be connected with RGC axons. We found elon-
gated astrocytes and star-shaped astrocytes similar to those first
described in primates by Ogden (1978). By contrast, in rat and mice
we only found star-shaped astrocytes as described in other species
(Fig. 5).
The numbers and distribution of astrocytes depends on the
density of the nerve fibres that they ensheath, as confirmed by
confocal microscopy and computer-assisted image reconstruction
of astrocytes in the vascularised retina of pigs, rats and mice
(Rungger-Brandle et al., 1993), and in the disposition of astrocytes
between species (Fig. 5). For example, in the fully vascularised
retina of cat, astrocytes are distributed throughout the inner retina
in a pattern that closely mirrors that of the axon bundles (Karschin
et al., 1986). The reconstruction of images from retinas stained for
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
Fig. 4. Astrocytes. (A) Minipig retinal section. RGCs are labelled with antibodies
against ßIII Tubulin (green), Müller cells and astrocytes are labelled with antibodies
against GFAP (red) and the nuclei are stained with DAPI. Note that the astrocytes follow
the blood vessels from the RGC layer to the outer nuclear layer (ONL). (B) Confocal
image of a rat whole-mount rat retina where astrocytes were labelled with antibodies
against GFAP (green) and aquaporin 4 (AQ4, red) (C) Rat glia primary culture. Astro-
cytes are indicated with an arrowhead, while the other cells are Müller cells with a
larger nuclei: vimentin labelling (green) and nuclei are stained with DAPI. Scale
bar ¼ 50 mm.
GFAP (astrocytes) allowed us to visualize the ensheathing of blood
vessels (Figs. 4B and 5A), whereby the vitreal and lateral sides of
most blood vessels are in close apposition to the vitreal surface of
the retina. In addition, individual astrocytes can be seen to extend
their processes into the vessel wall in such confocal images, espe-
cially in pig retinas, and while in some instances astrocytic pro-
cesses extend to the vitreo-retinal surface, in other cases astrocytes
remain parallel to RGC axons. It is noteworthy that astrocytes
communicate by gap junctions, and that both vitreous and blood
vessels can serve as a “sink” for Kþ (Newman et al., 1984).
2.2.2. Astrocyte markers
Astrocytic process envelops blood vessels as seen in electron
microscopy, with apparently no perivascular space between the
adventitia of the vessel wall and the glial cells (Hogan and Feeney,
1963; Hogan et al., 1971). GFAP is the major constituent of the
astrocytic intermediate filament (Bignami et al., 1972), and it rep-
resents a reliable marker to identify and localise astrocytes in the
mammalian retina. Under normal circumstances macroglia and
mammalian Müller cells contain significant amounts of GFAP and
thus, astrocytes must be classified as one of two distinct types in
the retina by attending to other molecular markers: type I astro-
cytes express GFAP and connexin-43. Whereas type II astrocytes
7
express GFAP but not connexin-43. Type I astrocytes can be further
divided into type Ia and type Ib, the former present in the optic
nerve head while type Ib astrocytes are present in the ILM of the
retina (Hernandez et al., 2000). Interestingly, both types are
quiescent astrocytes and they only become reactive in response to
certain stimuli, including elevated intraocular pressure, excitotox-
icity and retinal ischemia (Hernandez et al., 2008). When astrocytes
become reactive they hypertrophy and enlarge their soma, they
develop thicker astrocytic processes and their GFAP-
immunoreactivity is enhanced.
2.2.3. Astrocytes main functions
Despite our increased understanding of the function of inter-
mediate filaments (IFs) in astroglial cells (for a review, see Pekny,
2001), the role of astroglial IFs in maintaining the mechanical
integrity of these cells and that of the CNS tissue remains unclear.
As the main producers of VEGF (vascular endothelial growth factor)
during both normal (Stone et al., 1995) and pathological (Ozaki
et al., 2000) vessel formation, astroglial cells are strongly impli-
cated in retinal vascularisation. However, the vascular system de-
velops normally in GFAPe/e;Vime/e mice, and VEGF RNA is
expressed at normal levels in both ischemic and control GFAPe/
e
;Vime/e retinas, indicating that the absence of IFs in astroglial cells
does not affect their role in retinal vascularisation, either in healthy
or pathological conditions. The absence of IFs in astrocytes does not
impede the production of chondroitin sulphate proteoglycans, yet
it might delay the onset of astrocyte maturation and prolong the
window for optic nerve regeneration in GFAPe/e; Vime/e mice.
These data support the hypothesis that mature astrocytes represent
a key barrier to CNS axon regeneration in vivo (Cho et al., 2005) and
thus, astrocytes may play dual roles: one beneficial and another
degenerative.
Some of the beneficial roles fulfilled by astrocytes include
neurotrophic support, enhanced mechanical support for degener-
ating axons, and the maintenance of the blooderetina barrier. In
response to injury or disease, the same astrocytes express a number
of proteins that compromise the integrity of blooderetina barrier,
up-regulating the expression of various genes encoding cytokines,
chemokines and elements of the complement cascade, and pro-
moting retinal degeneration (Anderson et al., 1967; Kim et al.,
2006). Reactive astrocytes in the retina express increased levels
of MMP-9 and uPA, and accordingly, they degrade the extracellular
matrix associated with the ILM and they promote RGC death via
detachment-induced apoptosis (Zhang et al., 2004). In the different
sections that follow, we will refer to many other functions of as-
trocytes in more detail.
2.3. Microglial cells
Experimental evidence has confirmed the so called immune
privilege of the CNS on the basis of the existence of the bloodebrain
barrier (BBB), the absence of lymph or the lack of locally competent
macrophages. However, more recent research supports the notion
that the CNS establishes robust interactions with the immune
system and that the microglia fulfils a key role in controlling im-
mune responses (Carson et al., 2006). The CNS reacts through im-
mune responses that are both innate and adaptive, and that are
mediated by distinct populations of immune competent cells.
These cells may or may not share their embryonic origin but they
communicate through direct contact and paracrine interactions.
Microglia, astrocytes, infiltrating T-cells and macrophages form a
complex and pleiotropic defence network that secures and protects
the integrity of the CNS (Colton, 2009; Corraliza, 2014; Prinz and
Priller, 2014; Ransohoff and Brown, 2012).
Macrophages are a morphologically and functionally diverse
8 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
Fig. 5. Whole mount retinal explants. A, and C are control retinas. B and D are explants. A and B are pig retinas. C and D are mouse retinas. The astrocytes are labelled with
antibodies against GFAP (red in pig, A and B, and green in mouse, C and D). Note the changes in the astrocyte morphology between control and explant retinas. Scale bar ¼ 50 mm.
group of cells as a consequence of their embryonic origin, cellular
differentiation, tissue localization, and exposure to both internal or
external stimuli (Gordon, 2003; Gordon and Martinez, 2010; Prinz
and Priller, 2014). The macrophage family includes meningeal and
perivascular macrophages, blood circulating macrophages, den-
dritic cells derived from the bone marrow, and tissue resident
macrophages (Wieghofer et al., 2014). The microglia form a popu-
lation of resident macrophages within the CNS that not only has
immune activity but also, that participates in the development and
maintenance of neural networks, as well as in tissue homeostasis
(Fernandes et al., 2014; Gertig and Hanisch, 2014; Ransohoff and
Brown, 2012). Although the origin of these cells is controversial,
it is believed that primitive myeloid progenitors of the egg sac are
the precursors of these cells and not the bone marrow (Ginhoux
et al., 2010; Schulz et al., 2012), and during adulthood a popula-
tion of local progenitors ensures the continuity of the microglia in
the nervous system (Ajami et al., 2007). Microglia have a quite
specific morphology and physiology, very different from their
precursors, and they adapt to their neural environment to fulfil
their designated roles (Kettenmann et al., 2013).
As occurs with many advances in science (over time and
through persistence), the morphological and functional definition
of microglia has gradually been sculpted (Ginhoux et al., 2013). The
term “mesoglia” was proposed by Robertson (Ginhoux et al., 2013;
Kettenmann et al., 2013). Del Rio-Hortega in a conference delivered
in 1938 at the University of Oxford (UK), defined microglia as the
non-neuronal and non-astrocytic component of the “third
element”, of mesodermal origin, that can change its phenotype,
migrate and proliferate. He also separated microglia from the
oligodendroglia (Del Rio-Hortega, 1939), a characterization that can
still be applied today. Their plastic nature makes defining the
functional phenotypes of microglia difficult. However, it seems
clear that microglia can exist in different states of activation and
“quiescence”. Indeed, there seems to be a spectrum of intermediate
states or functional phenotypes that may explain their complex
physiology (Cherry et al., 2014). For instance, when using artificial
microstructured supports in vitro, these cells demonstrate a high
degree of plasticity in distinct extracellular milieus (Amadio et al.,
2013).
2.3.1. Origin, morphology and distribution of microglia
Several cell pools of myeloid origin and with common molecular
features populate the CNS, although they also exhibit important
differences relating to their origin and activity. Experiments in mice
confirmed that microglia originate in the yolk sac at an early stage
of embryonic development, between 8.5 and 9.5 days post-
fertilisation (dpf) (Ginhoux et al., 2010; Schulz et al., 2012). Spe-
cifically, they derive from erythromyeloid precursors (Kierdorf
et al., 2013). These stem cells undergo a sequential development
from stage A1 when they express the CD45 antigen to stage A2,
when they synthesize myeloid markers that include CX3CR1
(chemokine receptor 1) and CSF1R (colony-stimulating factor 1
receptor). Stage A2 cells migrate to the brain parenchyma with the
aid of specific matrix metalloproteinases (MMPs), where they
establish their final destination (Kierdorf et al., 2013; Prinz and
Priller, 2014; Prinz et al., 2011). By contrast, other macrophage
populations that inhabit the CNS have different myeloid origins,
which include the fetal liver, the aortaegonademesonephros re-
gion and the bone marrow (Kierdorf et al., 2013; Prinz and Priller,
2014).
Microglia colonise CNS structures during embryogenesis and in
the early postnatal period until they reach their positions in the
mature brain (Harry, 2013). Colonization occurs along different
migratory pathways, following glial structures, white matter net-
works or blood vessels (Rezaie and Male, 1999). In the CNS,
microglia are distributed widely in the gray and white matter, and
they present local singularities related to their function (Harry,
2013). The diverse morphologies of the microglia are related to
their activation states or functional phenotypes (Kettenmann et al.,
2011). Briefly, in the so called “resting” state they have a ramified
appearance with a small cell body and rather large nucleus. In the
“activated state” the cell morphology is more ameboid and they
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40 9

exhibit pseudopodia (Davis et al., 1994). In healthy conditions
microglia are found at variable densities in the different retinal
layers, including the NFL, ganglion cell layer (GCL), inner plexiform
layer (IPL), inner nuclear layer (INL) and OPL (Chen et al., 2014;
Cuenca et al., 2014; Garcia-Valenzuela et al., 2005; Noailles et al.,
2014; Santiago et al., 2014; Sobrado-Calvo et al., 2007). The cells
have a small ovoid-shaped cell body and multiple ramifications in
the GCL of the normal retina, and they are organized into a single
sheet that is distributed homogeneously (Garcia-Valenzuela et al.,
2005). In the optic nerve, microglia ramifications appear to be
orientated along the fibres. This orientation is disorganized in an
experimental model of diabetes (Chen et al., 2014), and the distri-
bution and organization of microglia within the retinal layers is
altered in a variety of pathological states, when changes in cell
shape can be observed (Cuenca et al., 2014; Kezic et al., 2013).
Moreover, when RGCs are labelled with a retrograde vital fluores-
cent dye in an experimental axotomy, microglia containing RGC
debris can be detected months after the injection of marker, indi-
cating both the phagocytotic capacity of microglia and their relative
longevity (Thanos et al., 1994).
2.3.2. Plasticity and functional microglia phenotypes
Microglia fulfil specialized immune tasks, responding to specific
stimuli by secreting cytokines and neurotrophic factors. In addition,
they establish a close relationship with neurons and other cells so
that they contribute to the remodelling of neuronal circuits, as well
as to the elimination of cell debris and deranged synapses. Micro-
glia also play a role in pathological events that affect the CNS
(Kettenmann et al., 2013; Prinz et al., 2011; Wieghofer et al., 2014).
Microglia exist in two separate morphological/physiological states,
namely “resting” and “activated” microglia (see for instance Colton,
2009; Kettenmann et al., 2011, 2013; Ransohoff and Perry, 2009). In
his early studies, Del Rio Hortega observed these two different
morphologies and he related them to the primitive invasion of
microglia (amoeboid) and the pool of settled cells in the mature
brain (branched and ramified: (Del Rio-Hortega, 1939). However,
although these two states serve as a reference point, the adaptable
and fine-tuned responses of microglia reinforce the existence of
several intermediate and fully functional phenotypes (Cherry et al.,
2014). One basic response of microglia to local insults depends on
the presence of extracellular ATP and it involves the specific
extension of microglial processes to the injured site (Davalos et al.,
2005). This morphologicalephysiological tour has been called the
“spider effect” (the spider in the middle of the web responds to
vibration caused by the “intruder” and moves to the site where the
prey is trapped, thereafter returning to its “vigilant” site in the
middle of the web: (Jonas et al., 2012). The concept of “resting” state
implies an “inactive” condition and it may lead to confusion, since it
suggests a passive behaviour that would only be converted to an
“active”, functional state under certain circumstances (for example,
infection, malignancy, trauma, neurodegeneration, etc…). Experi-
mental studies support the idea that even in healthy and homeo-
static conditions, microglia are responsive to changes in the
environment, and that they modulate numerous events by
secreting cytokines and other factors, and by interacting with other
glial cells and neurons (Kettenmann et al., 2011; Wieghofer et al.,
2014). Therefore, applying the term “surveying” instead of
“resting” to vigilant microglia seems to be more appropriate
(Santiago et al., 2014) (Fig. 6).
2.3.3. Representative molecular traits of microglia
Microglia produce many substances and they also have receptor
systems that process chemical signals from neighbouring or distant
cells. The complex physiology of microglia relies on their secretory

Fig. 6. Microglia. (A) Pig retina section where the microglia were labelled with antibodies against Iba-1 (red), astrocytes and Müller cells (green) were labelled with antibodies
against GFAP and the nuclei were stained with DAPI. (B) Amplification of A, where the filled arrowhead indicates a microglia cell around an astrocyte or Müller cell. (C) Amplification
of A where the empty arrowhead indicates the interaction between a microglial cell and a blood vessel. (D) Rat microglia primary culture where the mitochondria were labelled
with mitotrack (red) and the nuclei are stained with DAPI. Scale bar ¼ 50 mm.
10 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
capacity, and on the existence of multiple receptor systems for:
neurotransmitters and neuromodulators (e.g., dopamine, GABA,
glutamate, epinephrine/norepinephrine, cannabinoids, histamine,
angiotensin II, opioids, bradykinin, neurotrophins, acetylcholine,
etc…); cytokines and chemokines, including TNF-a (tumour ne-
crosis factor) or interleukins; complement; epidermal growth fac-
tor (EGF); Notch; macrophage colony-stimulating factor; peptides;
leukotrienes…etc. Microglia also express different transporter
systems for solutes and ions (Kettenmann et al., 2011).
Microglia communicate with other glial cells and neurons by
secreting factors that establish specific secretory signatures char-
acteristic of distinct functional or pathological states. The mole-
cules secreted by microglia and other glial cells (both proteinaceous
and non-proteinaceous) are defined as their secretome, and
establishing its composition may be essential to discover bio-
markers useful for diagnosis and prognosis, as well as in the search
for new therapies to alleviate different CNS pathologies. Here we
name just a few of the molecules that microglia are known to
secrete, either constitutively or in response to inflammation, injury,
CNS development and degeneration or homeostasis: cytokines and
chemokines, ROS (reactive oxygen species), nitric oxide (NO),
phospholipase A2 IIA type, BDNF (brain-derived neurotrophic fac-
tor), GDNF (glial cell line-derived neurotrophic factor), IGF (insulin-
like growth factor), FGF-2 (fibroblast growth factor 2) or LC1 (lip-
ocortin 1) (Jha et al., 2013). Cell contacts and soluble factors are key
elements to maintain the functional dialogue established between
the different cell types that populate the retina and they are char-
acteristic of a given activation state of microglia (Langmann, 2007).
A representative example of a neuronemicroglia interaction is the
FKN/CX3CR1 system (fractalkine or CX3CL1 and its G protein-
coupled receptor, CX3CR1). Fractalkine is a chemokine that may
exist as a membrane-bound protein or in a soluble form, and it is
present in neurons that bind to CX3CR1 expressed on microglia.
The CX3CL1 ligand modulates the activation of microglia in distinct
manners during homeostasis and in disease states (Sheridan and
Murphy, 2013). In the retina, the action of CX3CL1 depends on
the conversion of the membrane-bound and soluble isoforms, and
it participates in both neurogenesis and the response to cues that
trigger neurodegeneration (Zieger et al., 2014). Microglia also ex-
press adenosine receptors that regulate their activities both in
healthy and disease states (Santiago et al., 2014). Accordingly,
microglial receptor proteins appear as important targets for the
development of drugs to prevent or manage retinal neuro-
inflammation. The secretory capacity of microglia depends on non-
vesicular (transporters or ion channels) and vesicular mechanisms,
such as exocytosis and the release of extracellular membrane
microvesicles (EMVs) loaded with bioactive molecules (Prada et al.,
2013).
2.3.4. Identification of microglia
Due to the important role of microglia, both in homeostasis and
disease, it is of particular interest to develop tools to characterize
and identify these cells, as well as the molecular dialogue they
undertake in their multiple modulatory activities. This task is
difficult, particularly since this group of cells share molecular
identities with related cells of hematopoietic origin (Wieghofer
et al., 2014). The identification of cells is based on their localiza-
tion and morphology, as well as on the specific expression of sur-
face proteins and the functions associated to them. The
methodological approaches may involve the use of specific anti-
bodies to localize protein epitopes (immunocytochemistry, western
blotting…) or the manipulation of gene expression. The identity of
microglia can be assessed by using different protein markers (for
instance, IBA-1, ED-1, HLA-DR, CD-45…etc). In most cases, the
antibody markers (such as IBA-1 -ionized calcium binding adaptor
molecule 1- and ED-1 -monoclonal antibody against glycoprotein
CD68) can be used simultaneously to differentiate microglia from
other cell-related populations, including migrating macrophages
(Ellis-Behnke et al., 2013). Since all types of macrophages and
microglia express the F4/80 protein (a surface receptor of the EGF
family), this marker alone is not suitable to target microglia. His-
tochemical detection also offers advantages in some cases. For
instance, one histochemical method uses a tomato lectin with af-
finity for sugar moieties present on microglial and endothelial cells,
allowing the relationship of microglia with blood vessels to be
analysed (Villacampa et al., 2013). Microglia in different activation
states can be characterized using combinations of techniques
aimed at analyzing the secretory, proteomic and transcriptional
signatures (Glanzer et al., 2007): microarrays, real-time RT-PCR
(reverse transcription polymerase chain reaction), SELDI-TOF (sur-
face-enhanced laser desorption ionization-time of flight), two-
dimensional gel electrophoresis (2D-PAGE), mass spectrometry,
quantitative western blotting…etc.
Genetic manipulation serves to target microglia and has pro-
vided relevant tools to distinguish these cells from other myeloid
cells. Different approaches are available and include labelling
methods (where fluorescent reporter genes like GFP -green fluo-
rescent protein- or LacZ -beta-galactosidase- may help to monitor
the spatio-temporal expression of a given protein), as well as the
manipulation (activation and deletion) of genes that produce stable
transgenic animal lines (see Wieghofer et al., 2014 for a review). On
occasions it is difficult to separate macrophage populations and to
assign the specific involvement of each class to particular functions.
In a knock-in mouse model, where CNS microglia were labelled
with a GFP-tagged CX3CR1 and blood originated monocytes were
labelled with a RFP (red fluorescent protein)-tagged CCR2, the ge-
netic markers allowed “red” infiltrating monocytes and “green”
resident macrophages to be visualized (Saederup et al., 2010). This
method has also been applied to localize microglial populations
during development and at their final sites (Mizutani et al., 2012).
Based on the self-renewal capacity and the long lifespan of
microglia, a genetic system has been developed where the
expression of Cre recombinase (an enzyme that catalyzes site-
specific DNA recombination events) is controlled by the promoter
of a gene that encodes Cx3cr1 (Goldmann et al., 2013). This
experimental system allowed an in vivo animal model to be elab-
orated (Cx3cr1Cre mouse) in which conditional depletion of a
mitogen-associated protein kinase kinase kinase (MAPK3), TAK1
(transforming growth factor b-activated kinase 1), demonstrated
the direct participation of microglia in autoimmune inflammation.
This model opens new avenues to ascertain the role of different
genes expressed in microglia at specific time points or under spe-
cific circumstances (physiological or pathological). Since many
diverse techniques are available, it is important to be aware of the
potential and the limitations of each of these when designing ex-
periments to study the presence, morphology and function of
microglia.
3. Common functions of different glial cells in the retina
The three glial cell subtypes present in the retina share many
properties that while possibly redundant they have a preferential
hierarchy in which they are performed. There are certain functions
of retina glial cells that are more predominant in one cell type or
another. Thus, in the following sections we will review the most
recent advances related to this important issue and the role of these
glial cells in maintaining retinal homeostasis.
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
3.1. Formation of boundaries
Müller cells share many properties with astrocytes, including
the ability to ensheath axons, and to form glial boundaries between
the retina and the blood vessels (Hollander et al., 1991). However,
while Müller cells are formed in the retina, astrocytes migrate
independently of vessels and they are therefore in a position to
respond to environmental signals in avascular areas of the retina.
Their restriction to the inner layer of retina enables them to
respond to hypoxia in this region by expressing VEGF, thereby
inducing the formation of a superficial layer of blood vessels. By
contrast, Müller cells extend across all retina layers and cannot
respond in a layer-specific manner. The migration of astrocytes
across the surface of the retina therefore provides a population of
macroglial cells that is strategically located to sense hypoxia in the
inner layers of retina.
In addition to the glia limitans of the retina and vessels, astro-
cytes and Müller cells both contribute to the glial sheaths around
neurons. In particular, both astrocytes and Müller cells wrap gan-
glion cell axons into bundles, contributing to the glial convergence
on the initial segments and node-like axon structures, and they also
ensheath the somas of neurons in the RGC layer. Furthermore,
adherens junctions form between astrocytes, between Müller cells,
and between astrocytes and Müller cells, although not between
these cells and neurons or among neurons. These similarities point
to the possibility that astrocytes and Müller cells might act inter-
changeably in some processes, and we propose that they be
regarded as variants of macroglia. Quantitative differences between
astrocytes and Müller cells were noted in their ensheathment of
neurons. Specifically, the glial sheaths around the somas of RGCs
are predominantly formed by Müller cells (Fig. 3A,C), while the glial
processes attached to node-like specialisations of their axons are
mainly formed by astrocytes (Fig. 5A: Stone et al., 1995). In dog
retinas in particular we found that axons form packages that
interdigitate between the endfeet of the Müller cells in way similar
to the nests building (Fig. 2C).
One qualitative difference has been that in two glial cell classes
the gap junctions formed between astrocytes do not form between
Müller cells or between these two different types of cells. Thus, the
roles of the two types of glial cells, in terms of the information and
communication related to the cells that are ensheathed could vary.
Moreover, reactive astrocytes form a border between the lesion and
the surrounding tissues, and as resting microglia are located in
certain areas, close to where the retina is vascularised, they form an
imaginary fence that limits the penetration of any invader into the
retina.
3.2. Glia as a structural support for neurons
3.2.1. Viscoelastic properties
The biomechanical changes in the retina during its development
and in disease are very important, and they could be the underlying
cause of several conditions. During development and after the optic
vesicle has closed, the intraocular pressure and proliferation of
neuroblasts causes the primitive retina to stretch. After birth, the
retina continues to expand tangentially, stretching out from the
centre to periphery until the eye reaches a maximum size. During
this growth period the components of the retina must be elastic.
The retinal neurons are significantly stiffer than the glial cells, and
elasticity decreases from the inner to the outer retina. Moreover,
the two limiting membranes of the retina are stiffer that the rest of
the layers. While the Müller cell endfeet, nerve fibre bundles and
GC somata are stiff structures, the least elastic part of the retina is
the ONL as well as the inner and outer photoreceptor segments
(Franze et al., 2011; Lu et al., 2006). The elastic stiffness of retinal
11
Müller glial cells does not vary remarkably in the different
mammalian species studied so far (Lu et al., 2013), and their
viscoelastic properties appear to be similar in man, monkey and rat.
In general, their elastic behaviour dominates over their viscous
behaviour.
Müller cells act as mechanosensors, as they span vertically from
the inner to the outer retina. The Müller cells form stiff endfeet that
are rich in mechanosensitive ion channels, indicating that the
biomechanical responsive element of the retina is located in this
region. In addition to the different types of cells, the mechanical
properties of soft tissues like the retina mainly depend on extra-
cellular matrix components like collagen, elastin and pro-
teoglycans. One of the main functions of proteoglycans is to retain
water, which constitutes >70% of the retina. The presence of water
in tissues causes collagen fibres to separate, swell and become
disorganized, which significantly affects their relative stiffness and
tensile strength (Chen et al., 2009a).
The biomechanical scaffolding of the retina, as well as its
biochemical homeostasis, is maintained by Müller cells and astro-
cytes. These cells can alter their elasticity and stiffness by up- and
downregulating their IF proteins, the most common of which are
GFAP and vimentin, thereby altering both the tissue-wide and
cellular biomechanics of the retina (Bringmann et al., 2009a;
Lindqvist et al., 2010; Lu et al., 2011). In mice lacking GFAP and
vimentin, mechanical challenge revealed an exaggerated fragility of
Müller cells (Lundkvis et al., 2004). Müller cells are mechanically
heterogeneous in the lateral direction, although they seem to
display a mechanical homogeneity in the vertical direction. Due to
the distinct cytoskeletal organization in local regions of a Müller
cell, the regions closer to the endfeet, soma and processes can be
discerned by their mechanical properties (Lu et al., 2006). This local
variation in the mechanical compliance of a single Müller cell is
likely to determine the mechanical diversity in the retina (Park and
Lee, 2013). Mechanosensory Ca2þ ion channels, such as TRPV4, are
known to be present on Müller cells, particularly on the Müller cell
endfeet at the inner retinal border. Thus, these channels may also
provide the retina with a sensor of biomechanical changes
(Ryskamp et al., 2011).
In normal situations, in addition to morphological alterations
due to glutamatergic neurotransmission, there are traction forces
on Müller cells in the retina derived from the vitreous body. In the
human retina, the vitreous body adheres to the retinal tissue at the
peripheral retina through the major superficial retinal vessels, optic
disc, perifovea and macula. At sites of vitreo-retinal attachments,
the basement membrane of the ILM can become thinner, and vit-
reous fibres can adhere directly to Müller cells (Schubert, 1989).
However, there is limited information regarding the biomechanical
properties of these cells (and of the retinal tissue). Experimental
approaches are limited as it is unclear whether retinal cells and the
tissue of laboratory animals suitably reflect human conditions (Lu
et al., 2013).
Astrocytes are abundant in the CNS, and their role as passive
nurturing cells has been broadened to a more dynamic function in
terms of their relationship with other glial cells and neurons.
Because of the specialized nature of astrocytes, their morphology
and their location in the retina, one might envision that astrocytes
in the retina may contain mechanical sensors. Accordingly, astro-
cytes might sense eye movements and other perturbations of the
retina, for example an elevation of the intraocular pressure. Little is
known about the indicators of the mechanical state, however it has
been proposed that astrocytes at the optic nerve head could induce
astrocytic signalling by releasing ATP (Barres and Dowling, 2010). If
this was to occur, ATP release could potentially stimulate microglial
cells to migrate to the nerve head, activating astrocytes to become
reactive and influence neurons or blood flow (Barres and Dowling,
12 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40

Fig. 7. Changes in the distribution of BDNF and p75 receptors in control rat retinas and following ischemia. (A, C, E, G) Retinal sections where RGCs were labelled with antibodies
against BDNF. (B, D, F, H) Müller cells labelled with antibodies against p75 receptor. (A, B, E, F) are control retinas and (C, D, G, H) are the contralateral retinas where the ischemic
insult was maintained for 1 h, followed by 2 (C and D) or 24 h of reperfusion (G and H). Note the increase in immunofluorescence staining within the ganglion cell layer of C
compared to the control (white arrows) A, and the decrease in labelling in G as compared to E. Note the loss of the layering of Müller cells stained with antibodies against p75 in H
compared to F. Scale bar ¼ 50 mm.

2010). Astrocytes respond to mechanical forces by triggering
reactive gliosis, which might perturb their vasodilatory role by
disrupting their vessel contacts and interfering with their gap
junction coupling. Alternatively, this response could perturb the
ionic homeostasis, since astrocytes are also vulnerable to neuronal
death (Cahoy et al., 2008; Foo et al., 2011).
3.2.2. Architecture-mechanical forces
The axons within the retina of all species are organized radially
in order to converge at the optic nerve head, yet there are impor-
tant differences in the way that astrocytes are distributed within
the retina. While in the pig, minipig and human, astrocytes are
organized in two levels one more vitreal and another more internal.
The more vitreal astrocytes have a parallel disposition that can be
observed in flat mount retinas (Fig. 5A), while in rat and mice, the
distribution of astrocytes is more stellate appearance and their
extensions do not run parallel to the optic nerve (Figs. 4B and 5C).
Astrocytes can modify their distribution in response to local
changes and thus, after axotomy and explant culture the pattern of
astrocytes observed in flat mount retinas is lost within a few days. It
is possible that astrocytes sense the damage to the axons and thus,
they tend to lose their contacts and redistribute within the retina.
In mice and rats, astrocytes became hypertrophic and assume a
stellate shape, as observed in glaucoma (Fig. 5).
Müller cells also sense the changes that take place in the inner
and outer retina (see below, “glaucoma and retinitis”). Müller cells
modulate neuronal activity by regulating the concentration of
neuroactive substances and potassium in the extracellular space
through a variety of transport processes (Newman and
Reichenbach, 1996). This activity of Müller cells is particularly
important in ischemia when there is a massive release of
transmitters from neurons (Hossmann, 1993). The plastic changes
of the Müller cells take place over a relative short timescale (Fig. 7).
The three distinct layers of immunoreactivity originating from the
extensions of the Müller cells, that are present in the IPL of control
retinas, was no longer evident, rather, it appeared as a single thick
layer after ischemia (Fig. 7F, H).
The communication between Müller cells and RGCs remains
unknown, although we did observe changes to Müller cells in ret-
inas from animals with experimental glaucoma or axotomy
(Hernandez et al., 2009).
3.3. Volume regulation
The regulation of cell volume is of great importance to avoid
changes in neuronal excitability resulting from a decrease in the
volume of the extracellular space. Cell swelling and subsequent
Regulatory Volume Decrease (RVD) is accompanied by trans-
membrane potential (Vm) depolarization followed by repolariza-
tion. However, this RVD response depends closely on the
composition of the extracellular media. Although Kþ and Cl
channels play an important role in the RVD response of Müller cells,
their contribution is evident only if a significant driving force for
KCl efflux is present (Ferna
ndez et al., 2013). The changes in volume
of glial cells are also associated to the viscoelastic characteristics of
the retina.
It has been suggested that Müller cells possess endogenous cell
volume-regulatory mechanisms that are involved in the activity-
dependent regulation of the volume of the extracellular space
(Vogler et al., 2013). In addition to Kir (inwardly rectifying potas-
sium) channel mediated cell volume regulation, Müller cells
possess receptor-mediated mechanisms that prevent hypoosmotic
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
swelling (Uckermann et al., 2006; Wurm et al., 2008a). Thus,
pharmacological blockade of Kir channels causes swelling in brain
astroglial somata and processes, yet it has no effect on Müller cells.
The blockade of Kir channels in hypoosmotic conditions induces
immediate swelling of Müller cell somata but no additional
swelling in somata of brain astrocytes (Hirrlinger et al., 2008).
Water accumulation in retinal neurons and glial cells is a patho-
genic factor involved in retinal degeneration under ischemic-
hypoxic and inflammatory conditions. Nerve growth factor (NGF)
prevents the osmotic swelling of Müller cells by inducing auto-
crine/paracrine FGF signalling, as well as indirectly controlling the
swelling of bipolar cells by inducing a release of cytokines from
Müller cells (Garcia et al., 2014).
Müller cells of the rodent retina possess a volume-regulatory
glutamatergicepurinergic signal transduction cascade. Glutamate
induces the swelling of inner retinal neurons and shrinkage of the
extracellular space. Retinal glial cells rapidly and significantly
change their shape when glutamate is applied to retinal whole
mounts, and this effect is induced by glutamate-evoked neuronal
cell swelling (Uckermann et al., 2004; Wurm et al., 2008b). Müller
cells adapt their morphology to the increased size of activated
retinal neurons by extending and thinning the inner stem process.
This response is due to the opening of potassium and chloride
channels, whereby the ion efflux compensates for the osmotic
gradient across the Müller cell membrane and prevents cellular
swelling (Uckermann et al., 2006). The glial water channel
aquaporin-4 (AQP4) is implicated in the control of ion and osmo-
homeostasis in the retina. The water flux through AQP4 is
involved in the rapid volume regulation of retinal Müller cells in
response to osmotic stress and deletion of AQP4 results in an in-
flammatory response of the retinal tissue (Pannicke et al., 2010).
Müller cells can maintain the integrity of the blooderetina
barrier and clear metabolic waste by regulating the homeostasis of
extracellular pH and Kþ ions. To avoid high Kþ levels that might
induce depolarization of retinal neurons, Müller cells take up
excess Kþ from the extracellular space (especially in the synaptic
layers of the retina), and release it into the blood and vitreous
humour (Reichenbach and Bringmann, 2013; Reichenbach et al.,
2007). Many of the homeostatic functions of Müller cells,
including spatial potassium buffering and neurotransmitter uptake,
depend on the negative membrane potential (around 80 mV)
established by the ample expression of Kir4.1 in these cells, which
can regulate and compensate the osmotic conditions (Bringmann
et al., 2006; Kofuji et al., 2000). Kir 2.1 and Kir 4.1 channels were
recently studied under hypoxic conditions, which produced no
significant changes in their mRNA expression (Kang et al., 2013).
Adenosine might upregulate the expression of Kir 4.1 channels
while it only weakly affects the expression of Kir 2.1 channels
in vitro. Such upregulation of Kir 4.1 channels should accelerate
retinal Kþ clearance to prevent neuronal hyperexcitation and
excessive release of glutamate (Yu et al., 2012).
AQP4 are present in the plasma membranes of astrocytes that
are in contact with micro vessels (Fig. 4B), allowing them to regu-
late the extracellular space volume, waste clearance, potassium
buffering, cell migration and calcium signalling (Nagelhus and
Ottersen, 2013), playing an important role in water homeostasis
in the retina, moreover Müller cells play also a role in water
regulation.
3.4. Metabolism
3.4.1. Glucose
The retina exhibits metabolic peculiarities that are related to its
activity. Some specific characteristics of retinal oxidative and
anabolic metabolism are also found in tissues that manifest high
13
rates of proliferation (for instance, embryonic stem cells and
tumour cells: Ng et al., 2014). Glucose, obtained from external cir-
culation, from internal cellular glycogen stores or from lactate is the
primary metabolite used by retinal cells to produce energy, both in
aerobic and anaerobic conditions (Reichenbach and Bringmann,
2010).
Retinal cells also display specialised metabolic outcomes and
they have adapted to better serve cell house-keeping needs and
specific demands linked to processing visual inputs (photo-
transduction and neurotransmission). Within the retina, glial cells
and neurons orchestrate complex metabolic relationships
(providing nutrients and metabolites, responding to signals from
neighbouring or distant cells, or adapting the transcriptional ma-
chinery to supply the required metabolites) that fine tune their
metabolic activity to their needs. For example, Müller cells resist
hypoxia and low glucose environments by activating anaerobic
glycolysis and by oxidizing alternative substrates, such as lactate,
glutamate or glutamine in order to obtain energy in the form of ATP
(Kitano et al., 1996; Reichenbach and Bringmann, 2010; Stone et al.,
1999; Winkler et al., 2000). Compared with other tissues, the retina
consumes more ATP (Hurley et al., 2014) and it undergoes an
elevated proportion of aerobic glycolysis, the so-called Warburg
effect (glucose is preferentially converted into lactate, instead of
carbon dioxide and water by oxidative phosphorylation, despite the
presence of oxygen). The Warburg effect favours rapid ATP pro-
duction when glucose is converted into pyruvate and then fer-
mented into lactate to regenerate oxidized NAD (nicotine adenine
dinucleotide), and it may procure the energy needed for continuous
rhodopsin production or for other processes (Casson et al., 2013; Ng
et al., 2014).
A few examples confirm that retinal cells configure a metabolic
dialogue based on the expression and distribution of enzymes and
metabolic carriers. Retinal cells also exhibit different energy
metabolic profiles (for instance, glia, RGCs, photoreceptors) and a
single cell may have different energy demands in different com-
partments (as is the case for RGCs) while still maintaining the in-
ternal homeostasis (Yu et al., 2013). Glucose and lactate are not the
only primary metabolites catabolised to attend to the energy and
vegetative requirements of the retina. Retinal pigment epithelium
(RPE) cells metabolise fatty acids from phagocytised photoreceptor
outer segment membranes by beoxidation. Also, RPE cells produce
b-hydroxybutyrate from fatty acids (a ketone body) that is further
incorporated into the TCA (tricarboxylic acid cycle) by retinal cells
to obtain ATP, amino acids or other metabolites (due to the
amphibolic properties of TCA). Müller and microglial cells also
establish important metabolic relationships, both in functional and
diseased retinas. In physiological conditions, Müller cells release
ATP to the extracellular space that is used by microglia to fuel their
motility (Wang and Wong, 2014). Accordingly, an effective oxida-
tive and biosynthetic dialogue is established between retinal cell
components (Adijanto et al., 2014).
Among the well-defined house-keeping activities of astrocytes,
they provide neurons with essential metabolites (e.g., glucose), and
they take up Kþ and neurotransmitters from the extracellular space.
Although it was suggested that astrocytes can convert glycogen to
glucose, it is not known under what conditions this occurs in vivo.
Although glucose production via the G6Pase complex in the
endoplasmic reticulum is theoretically possible, other fates of G6P
in the cell include lactate-energy production via glycolysis and
entry into the pentose phosphate pathway. Also, there is contro-
versial evidence that confers a glucose export capacity on astro-
cytes per se. The metabolic relationship between different retinal
cell populations includes many molecules and reactions (Fig. 8),
and their detailed description is beyond the scope of this work. The
reader is referred to some recent works that deal with this matter in
14 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
Fig. 8. Diagram summarizing the main interactions of glial cells with neurons. Scheme showing glucose metabolism, Kþ homeostasis, H2O, glutamate (Glu) to glutamine (Gln)
metabolism, the secretion of trophic factors and interleukins: A (astrocytes, in green), B (bipolar cells), G (ganglion cells), M (Müller cells, in blue), Mi (microglia, in red), Ph
(photoreceptors).
more detail (Hosoya and Tachikawa, 2012; Hurley et al., 2014; Ng
et al., 2014; Reichenbach and Bringmann, 2010; Sanderson et al.,
2014; Yu et al., 2013).
3.4.2. Lipid metabolism
Anabolic activities in photoreceptors are primarily directed to-
ward the synthesis of outer segment discs, a highly membranous
compartment of the photoreceptor and the site of the photo-
transduction cascade. The retina has the capacity to both synthesize
cholesterol de novo and to take up lipids from the blood (Fliesler
and Keller, 1997). Circulating low-density lipoproteins (LDL) enter
the retina via LDL receptors on RPE and Müller cells (Tserentsoodol
et al., 2006). There is a lipid shuttle from Müller cells to neurons to
meet the need of neurons for lipids, especially to maintain the long
RGC axons and the photoreceptor outer segments, as well as for
synapse formation (Mauch et al., 2001). Müller cells and astrocytes
synthesize ApoE and ApoJ which are assembled into cholesterol-
rich lipoprotein particles that are thought to be secreted into the
vitreous humour. From there the particles may be taken up by RGCs
and transported centrally within the optic nerve. Although the
exact mechanism involved in this uptake is still unclear
(Amaratunga et al., 1996), the implication of Müller glia in the
intraretinal transport of lipids dependent on high density
lipoprotein-like particles has been proposed (Tserentsoodol et al.,
2006).
Docosahexaenoic acid (DHA) is a trophic factor required by
photoreceptors for their disk membranes, and it drives several ac-
tivities in the retina that can help protect photoreceptor cells from
regular exposure to photooxidative damage. As a precursor for the
neuroprotectin, NPD1, DHA helps to upregulate anti-apoptotic
genes of the Bcl-2 family, while downregulating pro-apoptotic
and pro-inflammatory genes (Bazan, 2007). Müller cells take up
DHA (Gordon and Bazan, 1990), incorporate it into glial phospho-
lipids and channel it towards the photoreceptors (Politi et al., 2001).
Müller cells also express fatty acid-binding and -transfer proteins
for the transport of DHA and other fatty acids (Deguchi et al., 1992;
Kingma et al., 1998). A potential mechanism for the anti-
inflammatory effects of DHA in diabetic retinopathy was recently
suggested, whereby microglia recruit receptors into lipid rafts
(Wang et al., 2015).
3.5. Extracellular matrix generators
The extracellular matrix (ECM) provided by macroglia is an
important source of supporting and signalling factors in the retina
and it contributes to the ILM, which is the boundary between the
retinal layers and the vitreous humour. ECM components are key
mediators of glial activation, and they have the capacity to evoke
both regenerative and degenerative effects on RGCs. The ECM
produced by glia is altered during reactive gliosis, with many
studies reporting a dramatic increase in the production of ECM
components and the re-expression of extracellular glycoproteins
that are down regulated after development. The following ECM
components have been identified in the healthy adult mammalian
retina, unless otherwise specified.
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
3.5.1. Laminin
Laminins are a family of glycoproteins that are a major con-
stituent of the ILM (Kohno et al., 1987), and the specific laminin
chains expressed in the retina are now starting to be elucidated.
Laminin acts to anchor cells to the ECM but it also has important
signalling functions transduced via transmembrane receptors like
integrins and dystroglycan. In the adult human retina, laminin g1
has been detected at the ILM of retinal astrocytes, and laminins a4,
b2 and g3 have been associated with Müller cells in the GC layer.
Other laminin chains present in the ILM that may be produced by
retinal macroglia include laminins a1, a5 and b1. The specific het-
erotrimeric complexes of these laminins in the retinal ECM have
not been fully determined (Libby et al., 2000). RGCs express laminin
chains a2 and b1 in the adult mouse retina, which suggests that
they contribute to their own survival (Morissette and Carbonetto,
1995; Sarthy and Fu, 1990).
3.5.2. Collagen
Collagen is another glycoprotein produced by glia and a con-
stituent of the retinal ECM Collagen types I and IV was identified in
the ILM of the human adult retina (Jerdan and Glaser, 1986).
Collagen type II was not found in the ILM but found in small
“packages” in the vitreous humour, closely associating with Müller
cell endfeet perhaps as a result of ECM remodelling (Ponsioen et al.,
2005). Furthermore, immortalised human Müller cells express
collagens Types I-VII, IX and XI (Ponsioen et al., 2008). Collagen-
XVIII was also detected at the ILM of the adult human retina.
Collagen XVIII is a basement membrane heparin sulphate proteo-
glycan (HSPG) that can be cleaved to form endostatin. Endostatin is
a broad spectrum inhibitor of angiogenesis and it may interfere
with the pro-angiogenic action of growth factors like basic FGF
(bFGF/FGF-2) and VEGF. This suggests that the expression of Type
XVIII collagen is related to retinal vascularisation in which astro-
cytes play an important role (Keenan et al., 2012).
3.5.3. Vitronectin
Vitronectin, an extracellular glycoprotein that can bind to many
components of the ECM and to integrins (specifically integrin
avb5). Vitronectin is expressed in the RGC layer of adult human and
mouse retinas, and it is up-regulated, along with its integrin re-
ceptor, following retinal damage (Anderson et al., 1999; Wang et al.,
2006). While vitronectin is expressed by RGCs, it is not expressed
by astrocytes or Müller cells (Anderson et al., 1999).
3.5.4. Fibronectin
Fibronectin is expressed by retinal astrocytes in neonatal rats
and it plays an important role in angiogenesis (Jiang et al., 1994).
Fibronectin was also shown to be produced by cultured adult
mouse retinal astrocytes (Scheef et al., 2005), although there are
inconsistent reports regarding fibronectin expression in the adult
retina and it remains unclear whether or not it is found in the ILM
of adult retina of different mammalian species (Chen et al., 2009b;
Deeg et al., 2011; Hiscott et al., 1992; Kohno et al., 1987; Ljubimov
et al., 1996). Fibronectin has an important developmental role in
vascularisation (Jiang et al., 1994), yet its function in the adult retina
requires further study.
3.5.5. Tenascin
Tenascins are extracellular matrix glycoproteins with adhesive
and counter-adhesive properties that can bind to a multitude of
associated ECM proteins and cell surface receptors, including
members of the integrin family (specifically a8b1 a9b1 avb3 and
avb6: Morrison, 2006). Tenascins can be cleaved by matrix metal-
loproteinases (MMPs) and serine proteinases, which alter their
binding capacities. Astrocytes from the adult rat optic nerve that
15
are unsupportive of RGC neurite outgrowth are reportedly tenascin
negative (although Tenascin C -TN-C- was not assessed), whereas
neonatal astrocytes that do support RGC outgrowth are found to
express tenascin (Bahr et al., 1995). A lack of tenascin expression
has ben proposed to contribute to the failure of adult CNS regen-
eration (Bartsch et al., 1992). More recently, TN-C upregulation was
seen in optic nerve head astrocytes after experimental injury in rats
(Johnson et al., 2007) and in glaucoma patients (Pena et al., 1999).
This suggests that tenascin is downregulated in cultured adult as-
trocytes that perhaps do not reflect events in vivo.
Interestingly, TN-C dampens the reactivity of cultured human
astrocytes, transforming them to a quiescent phenotype, and glia
appear to produce their own TN-C to modulate their own reactive
phenotype in an autocrine manner (Holley et al., 2005). Perhaps,
the expression of TN-C during regeneration serves to maintain a
manageable level of reactivity in order to promote a less inhibitory
environment for RGC regeneration. TN-C expressed by astrocytes
also acts at the retinal optic nerve junction, where it serves as a
crucial molecular barrier preventing myelinating oligodendrocyte
precursor cells (OPCs) from entering the retina (Bartsch et al., 1994;
Morcos and Chan-Ling, 2000).
Cultured Müller glia from P3 mice promote RGC axonal
outgrowth and express the large TN-C isoform (Siddiqui et al.,
2009). Indeed, some isoforms of TN-C inhibit neurite extension
whereas others vigorously promote neurite outgrowth, depending
on their FnIII domains. The FNIII Domain D of TN-C is necessary to
stimulate retinal outgrowth on Müller glia cells (Siddiqui et al.,
2008). An increase in Müller cell proliferation has been observed
on stiff substrates and an upregulation of connective tissue growth
factor (CTGF) and TN-C on softer substrates (Davis et al., 2012).
Recently, Müller cells were shown to have the potential to re-enter
the cell cycle and differentiate into multipotent neuronal pro-
genitors that can regenerate cells in damaged areas. This can be
induced in vitro by exposure to FGF2. TN-C is important in this
process as it enhances Müller cell sensitivity to FGF2. Moreover, de-
differentiation was found to be impaired in TN-C knock-out mice
(Besser et al., 2012).
3.5.6. Proteoglycans
Chondroitin Sulphate Proteoglycans (CSPGs) are produced by
retinal glia and have both positive and negative effects on RGCs.
CSPGs are crucial mediators of axonal pathfinding during devel-
opment, yet they are known to be inhibitory to axonal regeneration
(Carulli et al., 2005). The CSPG, neurocan, is upregulated by Müller
cells in adult rat retina during reactive gliosis, suggesting that these
cells may not support RGC regeneration (Inatani et al., 2000). A
comparative study of the ECM produced by different glial cells
found that the ECM of Müller cells from neonatal mice express low
levels of CSPGs and high levels of growth promoting molecules like
laminin, fibronectin and TN-C. These Müller cells are a favourable
substrate for RGC axon outgrowth and when the production of
CSPGs is inhibited, axonal growth is enhanced. This suggests that
the ECM of neonatal Müller cells contains more stimulating than
inhibitory components and that it is a favourable substrate for RGC
axons (Siddiqui et al., 2009). These results highlight the need to
further analyse the ECM produced by adult retinal glia in order to
assess which growth promoting and growth inhibiting factors are
altered with respect to neonatal cells.
Heparan Sulphate Proteoglycans The ILM of the adult human
retina contains diverse heparan sulphate proteoglycans (HSPGs)
including perlecan, agrin and Collagen Type XVIII (Clark et al., 2011;
Keenan et al., 2012). HSPGs are crucial for axonal guidance during
retinal development (Chai and Morris, 1999), although their role in
the adult retina is not well understood. In mature tissue, sulphated
proteoglycans are crucial for the correct function of many signal
16 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
transduction pathways and for the binding of a multitude of mol-
ecules, including growth factors (e.g., VEGFs and FGFs), growth
factor receptors, chemokines, matrix molecules, proteases and
enzyme inhibitors. For this reason, it would be interesting to
explore the roles of HSPGs in relation to mature RGCs since there
may be multiple signalling pathways that are dependent upon this
type of regulation.
3.5.7. Intermediate Filaments (IFs)
Following CNS injury, astrocytes and Müller cells upregulate the IF
proteins GFAP, vimentin and nestin, which alters their physical
properties and makes the cells more rigid. This allows them to
establish a glial scar that is a physical and biochemical barrier to
infection, as well as a barrier to regeneration. The upregulation of IFs
also increases contact between cytoskeletal components and the ECM,
enabling the cells to initiate rapid cytoskeletal alterations. Astrocytes
and Müller cells have a polarised morphology (Enger et al., 2012) and
the loss of this polarity may be one factor that contributes to their
reactivity as they lose the crucial connection with the ECM.
In the retina, astrocytes and Müller cells express GFAP and
vimentin at detectable levels in normal adult tissue and both are
dramatically upregulated following damage to the retina (Lewis
and Fisher, 2003). It is unclear if GFAP is expressed by unreactive
Müller cells since while there are reports that it is only expressed at
times of stress (Kim et al., 1998; Xu et al., 2009) it has also been
detected in Müller cell processes of healthy retinas (Erickson et al.,
1987). Increased expression of GFAP and vimentin increases the
stiffness of the cells, which does not encourage axonal regeneration
because growing neurites prefer soft substrates (Bringmann and
Wiedemann, 2012). There is also evidence that retinal astrocytes
in zebrafish express GFAP weakly and rather, they rely on cyto-
keratin IFs to maintain their structural stability, which perhaps
produces a less rigid scar than in mammals (Koke et al., 2010).
There may also be alterations to intrinsic cell receptors in adult
RGCs that make them unresponsive to extracellular guidance cues
since they are less capable of extending processes through 3-
dimensional cultures of astrocytes than embryonic RGCs, suggest-
ing that their cellecell adhesion properties are significantly altered
(Fawcett et al., 1989).
Nestin is expressed very weakly in healthy tissues but it is
upregulated after retinal damage, for example in induced glaucoma
(Xue et al., 2006a), optic nerve transection (Xue et al., 2006b) or
after laser injury (Kohno et al., 2006). Nestin was recently shown to
be upregulated in a subpopulation of Müller/astrocytes in the RGC
layer in a rat model of Retinitis pigmentosa. Nestin is better known
as a stem cell marker and it is associated with proliferating cells,
such as stem cells and progenitor cells, and it was proposed that the
re-expression of nestin signifies the dedifferentiation of Müller
cells (Valamanesh et al., 2013). The upregulation of nestin by Müller
cells and astrocytes has also been reported following experimental
retinal detachment in the adult rat and where differential expres-
sion of nestin within the Müller cell population was observed (Luna
et al., 2010). Synemin is another IF protein that is upregulated in
both Müller glia and astrocytes and like nestin, it is similarly
downregulated after development and its re-expression in Müller
cells following injury may be an indicator of de-differentiation
(Luna et al., 2010).
In summary, the ECM substrate provided by confluent Müller
cells in vitro favours the growth and regeneration of RGCs (Garcia
et al., 2002). Moreover, the best conditions for RGCs involve a
combination of ECM plus secreted factors from Müller cells (Vecino
et al., 2015b).
3.5.8. N-Cadherin
N-Cadherin is essential for neurite outgrowth, as evident when
embryonic chick RGCs are cultured on rat cortical astrocytes
(Neugebauer et al., 1988). Similarly, N-Cadherin is required for
neurite outgrowth when in contact with Müller cells in vitro and
in vivo (Riehl et al., 1996). Retinal axons from embryonic mouse
explants express N-cadherin when cultured on astrocytes whilst
those from adult explants do not.
N-Cadherin fulfils crucial developmental functions in the em-
bryonic retina and it is downregulated in the adult retina (Bates
et al., 1999). However, N-Cadherin is upregulated during regener-
ation in the adult zebrafish retina (Liu et al., 2002). Thus, the
inability of the mammalian retina to re-express N-Cadherin
following injury may be one of the reasons that RGCs don't
regenerate, making N-Cadherin a potential therapeutic target.
3.5.9. Integrins
Integrins are membrane-spanning adhesion molecules that link
the intracellular cytoskeleton to the ECM. Integrins are composed
of a and b subunits, of which there are multiple types, and the
resulting heterodimers allow cells to respond to adhesion through a
number of different ECM ligands. Integrin-mediated cell-adhesion
allows cells to communicate with their environment and the sur-
rounding ECM, favouring alterations to the extracellular milieu to
be relayed back to the cell, which then alters its morphology
accordingly. Loss of the connection between the cell and the ECM,
for example by MMP induced proteolysis, is one way in which
retinal degeneration can progress. Based on current evidence,
integrin signalling in RGC-glia interactions is crucial for cell sur-
vival, process extension and the maintenance of correct retinal
function, although the promiscuity of integrin subunit hetero-
dimers combined with their temporal expression in the retina
make it difficult to draw definitive conclusions regarding the
functions of specific integrins. What is clear is that throughout
retinal development and during degeneration there is a strong
requirement for b1 integrin signalling to promote cell survival and
outgrowth. The importance of b1 class integrins in mediating RGC
neurite outgrowth in the developing retina has been well charac-
terised in the chick and mouse embryonic retina and the a-subunits
were found to be expressed in RGCs (Bosco et al., 1994; Bossy et al.,
1991; Bradshaw et al., 1995; Cann et al., 1996; Cohen et al., 1987; de
Curtis, 1993; Duband et al., 1992; Hall et al., 1987; Sheppard et al.,
1994).
Adult rat RGCs preserve the capacity to survive and regenerate
in the presence of different ECM components, such as laminin,
fibronectin, collagen I and collagen IV, demonstrating the tremen-
dous plasticity of adult RGCs to reprogram their ability to express
different integrins depending of the substratum in which they grow
(Vecino et al., 2015b). It is clear that RGCs are in contact with
different ECM components produced by different glial cells within
the retina. As the axons exit the optic nerve they come into contact
with the ECM components of the optic nerve head, lamina cribosa,
optic nerve axons and oligodendrocytes. Thus, RGCs must express
different sets of integrins in order to survive when in contact with
different substrata and glial cell types, preserving their capacity to
regenerate. We have described the beneficial effect of Müller cells
on the survival and the capacity of RGCs to regenerate neurites
(Fig. 9). Müller cells secrete beneficial factors but they are even
more beneficial when RGCs are grown on a confluent Müller cell
substratum (Garcia et al., 2002; Higginson et al., 2013). The bene-
ficial effects of Müller cells therefore appear to be due to secreted
factors and adhesion molecules present on these cells.
Adult human Müller cells express a1, a2, a3 and b1 integrin
subunits in culture (Guidry et al., 2003) and cultured rat Müller
cells from P3 rats strongly express the b1 integrin subunit (Siddiqui
et al., 2009). Multiple integrin heterodimers are expressed in as-
trocytes and the expression of a2, a3, a6, b1 and b4 integrin
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
Fig. 9. Effect of Müller cells on RGCs in vitro. (A) Rat RGCs cultured on confluent pig retinal Müller cells monolayer. (B) RGCs incubated directly on the laminin-poly-lysine substrate.
RGCs were labelled with antibodies against-bIII Tubulin (green) and the nuclei were stained with DAPI (blue). Note that there are more RGCs when grown on Müller cells, and with
longer neurites. Scale bar ¼ 50 mm.
subunits by astrocytes of the adult human optic nerve head has
been reported (Morrison, 2006). Thus, based on the resultant het-
erodimers, we can predict that laminin and collagen are important
ECM components in this structure. In terms of RGC-astrocyte in-
teractions, whilst there could be bi-directional integrin communi-
cation in both cells, the primary role of astrocytes in this context is
likely to be the provision of binding partners for the integrin sub-
units expressed by RGCs in order to relay survival signals.
3.6. Regulation of neuronal activity
Glial cells are classically viewed as passive participants in syn-
aptic function. However, there is much evidence that there is a
dynamic two-way communication between glia and neurons at the
synapse (Barres, 1991). Neurotransmitters released from pre-
synaptic neurons evoke increases in Ca2þ in adjacent glia. Acti-
vated glia then release transmitters, including glutamate and ATP,
and these glio-transmitters feedback onto the pre-synaptic termi-
nal to either enhance or supress further neurotransmitter release.
Transmitters released from glia can also directly stimulate post-
synaptic neurons, producing either excitatory or inhibitory re-
sponses. Müller cells envelop synapses in the retina, like Bergmann
glia in the cerebellum or Schwann cells at the pre-synaptic
neuromuscular junction. Thus, the precise shaping of synaptic ac-
tivity depends upon the kinetics of both the pre-synaptic neuro-
transmitter release and transmitter re-uptake. In the retina,
photoreceptor cells, neurons and macroglial cells express high-
affinity transporters for neurotransmitters, all contributing to
synaptic function in the retina.
As previously indicated in this review the role of microglia is
mainly related to their macrophage action during damage and to
their capacity of secreting chemical signals that contribute to
inflammation, cell death and cell survival. However, microglia also
have essential activities that control synaptic connections and
synaptic activity in the healthy brain, both during development and
in adulthood (Schafer et al., 2013). Microglia contact synaptic ele-
ments, dendritic spines and peri-synaptic astrocytes, through
processes that extend and retract dynamically (Tremblay et al.,
2010). The release of microglial factors influences both neuro-
transmission and synaptic plasticity (Bilimoria and Stevens, 2014;
Schafer et al., 2013). Microglia modify the number of synapses in
different circumstances and they directly eliminate specific syn-
aptic contacts or those in close collaboration with astrocytes
(Miyamoto et al., 2013). One key candidate that seems to play an
important role in synapse elimination/remodelling is the classical
complement cascade (Mastellos, 2014; Stephan et al., 2012; Stevens
et al., 2007). For instance, the remodelling of synapses during the
17
development of the visual system depends on the integrity of the
complement cascade, given that genetic deletion of important
components of this protein family block the capacity of microglia to
properly remove specific synapses (Schafer et al., 2012). Indeed,
astrocytes induce the expression of complement elements in the
CNS and they act in conjunction with microglia that eliminate
complement tagged synapses during synapse pruning (Stephan
et al., 2012). Astrocytes also engulf and eliminate synapses
through a mechanism independent of complement activation, both
during development and in adults (Chung et al., 2013). Other mo-
lecular mechanisms that trigger the phagocytotic intervention of
microglia involve the major histocompatibility complex (MHC class
1), glutamate receptors and ATP signalling, etc (Miyamoto et al.,
2013).
3.6.1. Gamma-Aminobutyric acid (GABA)
In addition to GABA and Glutamate, Müller cells express, take up
and exchange various neurotransmitters (Bringmann et al., 2013).
In the outer retina, Müller cells are responsible for all GABA
removal, whereas they share that task with amacrine cells in the
inner retina (Marc, 1992). Astrocytes express a wide variety of
neurotransmitter receptors (Porter and McCarthy, 1997) and glia
responses to neurotransmitters were initially characterized using
cultured astrocytes as a model system. The release of several
different neurotransmitters from active neurons can stimulate glia
in intact tissue preparations, evoking increases in glia calcium.
These responses are mediated by several glutamate receptors,
GABAB receptors and muscarinic ACh receptors (Kang et al., 1998;
Pasti et al., 1997). Moreover, the capacity of glia to respond to
neuronal activity suggests that they might regulate local blood flow
in response to changes in neuronal activity (Paulson and Newman,
1987), which would be crucial for retinal damage.
Transmitter release from neurons can stimulate glia and pro-
voke the release of glutamate, ATP and other neuroactive sub-
stances from glia. These gliotransmitters that are released by glia
can directly stimulate post-synaptic neurons, evoking either
excitatory or inhibitory responses in these cells (Newman, 2003).
However, whether such responses also take place in vivo remains
unclear. Another interesting question that must be addressed is if
the heterogeneity of glial cells and their actions influence the sur-
vival of different neurons, or if different neurons are responsible for
modulating resistance to damage.
3.6.2. Glutamate
Glutamate is the main excitatory neurotransmitter in the retina,
which is used in the transmission of visual signals by photorecep-
tors, bipolar and ganglion cells. In the outer retina, glutamate is
18 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
continuously released by photoreceptor terminals in the dark and
this release is inhibited by light. In the IPL, ON-bipolar cells release
glutamate during light exposure whereas OFF-bipolar cells release
glutamate in the dark (Bringmann et al., 2013). Müller cells are the
main elements responsible for the removal of excess glutamate
released by the RGCs. The Müller cells are interposed between the
vessels and RGCs, creating a barrier through which the Müller cells
provide nutrients and release essential substances to the RGCs
(Bringmann et al., 2009b). The major glutamate uptake carrier of
Müller cells is the electrogenic glutamate-aspartate transporter
(GLAST, excitatory amino acid transporter-1, EAAT1: Derouiche and
Rauen, 1995).
After EAAT-mediated glutamate uptake into Müller cells, gluta-
mate is converted to glutamine by glutamine synthetase (GS), and
subsequently, glutamine is transported back to the neurons for
glutamate re-synthesis (Danbolt, 2001). Glucose deprivation results
in an increased glutamate uptake as well as the upregulation of
EAAT1 and EAAT2 expression, indicative of a regulatory mechanism
that prevents excitotoxicity in neighbouring RGCs. Indeed, glutamate
uptake by Müller cells would appear to be essential to RGC survival
(Toft-Kehler et al., 2014). The transport process itself triggers a signal
transduction cascade that involves the activation of p60src, PI3K and
PKB (Akt) linked to extracellular Ca2þ entry, which not only promotes
translation by Ser-2448 phosphorylated mTOR but also, transcrip-
tional activity mediated by Ca2þ-dependent binding of inducible
transcription factors, such as AP-1. These results further support the
pivotal role of glia cells in glutamatergic transmission in the retina
(Lopez-Colome et al., 2012). Significantly, various receptor agonists,
including VEGF, erythropoietin and neuropeptide Y can induce the
release of glutamate from Müller cells, which induces ATP release
upon activation of mGluRs (Bruckner et al., 2012).
3.6.3. ATP and adenosine
Glutamate and ATP are candidates for neuron-to-glia signalling
associated with volume (Uckermann et al., 2006). Indeed, ‘patho-
logical’ osmotic swelling of retinal (Müller) glia in rat retinal slices
(evoked by the potassium channel blocker, barium chloride, by
inflammatory lipid mediators, or under pathological conditions like
diabetes and ischemia) can be prevented by pharmacological acti-
vation of purinergic P2Y and adenosine receptors (Uckermann
et al., 2006; Wurm et al., 2008a). Activation of purinergic re-
ceptors was recently concluded to be fundamentally involved in the
regulation of glial cell volume and thus, in the homeostasis of the of
the extracellular space mediated by these cells (Wurm et al., 2011).
Moreover, the inhibitory effects of VEGF and glutamate on the
swelling of mouse Müller cells is mediated by transactivation of
P2Y1R, as well as by intracellular calcium signalling through IP3R2
activation (Grosche et al., 2013).
Adenosine is a reactive metabolite involved in cellular
communication in some pathological states and in the eye, retinal
ischemia and/or hypoxia provokes an increase of adenosine. Four
adenosine receptor subtypes have been identified (A1, A2A, A2B
and A3: Kang et al., 2013). In the retina, adenosine suppresses
excitatory neurotransmission through various mechanisms,
including the inhibition of pre-synaptic voltage-dependent calcium
channels (Housley et al., 2009). Since adenosine is a downstream
mediator of the swelling-inhibitory actions of glutamate and ATP, it
is likely that the inhibitory effect of adenosine induces chloride
channel opening (Bruckner et al., 2012).
3.7. Synaptic pruning
Synaptic pruning refers to the sculpting that reduces and re-
models excess synaptic neuronal contacts to obtain functional
neuronal circuits, a process that takes place mainly during
development but that also occurs in the mature brain. Astrocytes
and microglia fulfil a key function in synaptic pruning through
different mechanisms that ultimately combine to conform a robust
neural network. Microglia employ their phagocytotic and secretory
activities to control the neural circuits in healthy brains (Bilimoria
and Stevens, 2014) In mouse, for example, microglia engulf syn-
apses during synaptic maturation given that proteins like PSD95 (a
marker of excitatory postsynaptic density) and SNAP25 (a pre-
synaptically localized protein) can be found inside microglial cells
(Paolicelli et al., 2011). Synaptic pruning is delayed in mice with
microglia lacking the chemokine receptor Cx3cr1, indicating that
their neuronal networks were immature (Paolicelli et al., 2011).
Astrocytes in both the adult and developing brain mediate the
elimination of synapses and therefore, they participate in the
reshaping of neural circuits. Astrocytes participate in the formation
of functional synapses within the CNS and they can control synapse
maintenance in vitro (Ullian et al., 2001). Glypican (HSPGs) 4 (Gpc4)
and 6 (Gpc6) are secreted by astrocytes, and they induce functional
synapses between purified RGCs (Allen et al., 2012) Indeed, the
application of Gpc4 to neurons increases glutamatergic neuro-
transmission. Astrocytes can also eliminate synapses through two
phagocytotic receptors, namely Megf10 and Mertk, and their ca-
pacity to engulf synapses depends on neuronal activity (Chung
et al., 2013).
The complement system is part of humoural immunity,
involving a group of proteins that participate in the elimination of
alien cells and debris. However, new functions of this tagging sys-
tem are observed in relation to neuronal differentiation and the
establishment of neuronal networks in the CNS (Mastellos, 2014).
Synaptic components tagged with proteins of the complement
system are eliminated by microglia and astrocytes, and they
collaborate in this process by secreting signals that induce the
synthesis of complement proteins by neurons (Stephan et al., 2012).
Selective elimination of synapses during post-natal development
requires the expression of C1q in cultured RGCs, the initiating
protein in the complement cascade (Stevens, 2008; Stevens et al.,
2007). Moreover, the absence of the C1q or C3 protein in mice
(downstream elements in the complement cascade) leads to
excessive retinal innervation of the LGN (lateral geniculate nucleus)
as a consequence of defective synapse pruning, and neural activity
regulates the engulfing capacity of microglia (Schafer et al., 2012).
Other phagocytotic mechanisms may be implicated in the process
of synaptic pruning and thus, further studies in other regions of the
CNS and in different experimental models will be needed to
ascertain the precise role of microglia and astrocytes in the
remodelling of synapses during development and in maturity.
Indeed, many questions are still unanswered and the role of glial
cells in CNS remodelling remains ill-defined (Schafer et al., 2013).
A close relationship exists between Müller cells and RGCs,
whereby glial cell processes often ensheath ganglion cell somata, as
well as some of the axonal, dendritic and synaptic regions of these
retinal neurons, an ensheathing pattern also common to astrocytic
processes (Hollander et al., 1991). This intimate association be-
tween glia and neurons is thought to allow glial cells to promote
synapse formation and it helps maintain neuronal function by
providing nerve terminals with energy substrates and neuro-
transmitter precursors (Pfrieger and Barres, 1996).
3.8. Immune responses
The retina is considered an immune-privileged tissue and
therefore, a multitude of mechanisms operate in the eye to limit the
harmful inflammation that foreign substances provoke. At the same
time, different mechanisms defend the eye from invading patho-
gens, with ocular cell surface and soluble factors maintaining the
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
eye's immunosuppressive nature. RGCs are thought to play a major
role in the recognition and subsequent clearance of pathogens
during infection, and they are believed to represent the first line of
defence in the retina. While microglial cells are mainly responsible
for these effects, astrocytes and Müller cells also collaborate in this
activity. Microglial cells are capable of modifying their positions in
order to maintain homeostasis, however, both astrocytes and
Müller cells can detect damage since both expand their projections
to the blood vessels while essentially maintaining their positions.
Different immune cells are situated at different locations within
the retina. Dendritic cells (MHC-IIþ33D1þ) are preferentially
located around the optic disc and the peripheral margin of the
retina (Xu et al., 2007). Moreover, their role in optic nerve and
retinal inflammation remains to be elucidated. Perivascular mac-
rophages play a critical role in retinal vascular inflammation and
they may also participate in neuronal retinal defence. Microglial
cells are the main innate immune cells in the retina and their
activation is tightly regulated by a number of inhibitory pathways,
including CD200/CD200R (Copland et al., 2010) or CX3CL1/CX3CR1
(Chen et al., 2012). Complement proteins are expressed in the
retina under normal physiological conditions (Luo et al., 2011) and
their expression augments in pathological conditions like inflam-
mation (Chen et al., 2010) and ageing (Chen et al., 2007). Comple-
ment activation is related to various retinal inflammatory and
degenerative diseases, and modulating these pathways may have
therapeutic applications.
Recent studies have shown that Müller glia actively participate
in retinal innate immunity, specifically in infectious conditions like
bacterial endophthalmitis, an activity that involves their Toll-like
receptors, phagocytosis, and the secretion of cytokines and che-
mokines. Müller cells also influence the adaptive arm of immunity
by inhibiting the proliferation of T-cells via membrane-bound
molecules. As a consequence of Müller cell activation, pro-
inflammatory cytokines and other potent mediators of inflamma-
tion are synthesised and released (Kumar et al., 2013).
Müller glia cells increase complement protein expression
(Kuehn et al., 2006). The complement protein C1q, an initiator of
the classical complement cascade, is upregulated in neurodegen-
erative diseases like Alzheimer's and amyotrophic lateral sclerosis
(ALS), as well as in rodent and human glaucoma (Stasi et al., 2006;
Stevens et al., 2007). The complement cascade has been implicated
in the normal elimination of synapses by RGCs during visual system
development in the DBA/2J mouse model of glaucoma (Howell
et al., 2007). Thus, it might be possible that complement protein
deposition occurs prior to significant loss of RGCs in the IPL. These
findings strongly suggest that a normal mechanism of develop-
mental synapse elimination is reactivated in the mature visual
system by the glaucomatous process, and that this process of syn-
apse elimination may well entirely drive glaucomatous neuro-
degeneration. Most of the secreted C1q is released by microglia,
which express C1q mRNA strongly. However, what communication
occurs between astrocytes and microglia, and the chronology of the
events that take place in the early stages of glaucoma remain un-
clear, although it is known that microglial activation is an early
event in this process (Bosco et al., 2008; Steele et al., 2006).
Müller cells produce different cytokines in response to different
stimuli. For example, IL-1b (interleukin-1b) and TNF-a are potent
inducers of IL-8 expression in Müller cells (Liu et al., 2014b), and IL-
1b and lipopolysaccharide (LPS) also provoke IL-6 production
(Yoshida et al., 2001). IL-6 is a key cytokine in the host's defence
against environmental stress, such as infection, inflammation and
injury (Mesquida et al., 2014). Indeed, the dysregulated and
persistent production of IL-6 has been implicated in the develop-
ment of various autoimmune and chronic inflammatory diseases,
as well as ocular disorders. These cytokines can stimulate
19
fibroblasts, endothelial cells and macrophages to produce chemo-
kines, promoting the recruitment of neutrophils and macrophages
to the retina, thereby resulting in further tissue damage and chronic
inflammation. It is now known how IL-6 expression is regulated in
Müller cells. These cells produce high levels of IL-6 in response to
IL-1b stimulation and activation of the p38MAPK/NF-kB signalling
pathway plays a predominant role in regulating IL-6 expression (Liu
et al., 2014c).
Some activities are common to the immune system and CNS: the
secretion of immunoregulatory cytokines; the response to cyto-
kines; and antigen presentation. These properties reflect the
physical contact between the two systems (microglia, astrocytes
and Müller cells presenting antigen to T cells) and their commu-
nication via soluble factors like cytokines. Indeed, cytokines
mediate a complex circuit of interactions, especially in the event of
lymphoid-mononuclear cell infiltration into the retina. The secre-
tion of interferon g (IFN-g) by infiltrating activated T cells could
initially induce astrocytes, Müller cells and microglia to express
class I and class II MHC antigens, as well as priming these cells for
subsequent cytokine production that would contribute to either the
initiation and/or propagation of immune responses. Müller cell-
mediated inhibition of T cell proliferation can be overcome by
treating Müller cells with glucocorticoids, presumably preventing
the expression of the Müller cell inhibitory factor (Roberge et al.,
1991), supporting a role for Müller cells as antigen-presenting
cells (APCs) in the retina. A number of inflammatory mediators in
the CNS act by ultimately suppressing immune responses, inhibit-
ing class I and II MHC expression, and dampening cytokine pro-
duction by glial cells, such as that of prostaglandins, IFN-a and IFN-
b, TGFb and neurotransmitter like norepinephrine. The induction
and ultimate downregulation of immune responses and cytokine
production within the retina depends on: a dynamic interaction
between a variety of peripheral immune cells and retinal cells; the
activation of these cells; the presence of cytokines with pleiotropic
effects; the concentration and location of these cytokines in the
retina; and the temporal sequence in which a particular cell re-
sponds to these cytokines. Thus, the ultimate outcome of immu-
nological and inflammatory events in the retina will be determined
in part by the interplay of these parameters (Benveniste, 1993).
3.9. Gliosis
In the retina, reactive gliosis alters the morphology of the retina
through hypertrophy, Müller cells and astrocytes provoking the
thickening and enlargement of processes. This response occurs in a
multitude of disorders like trauma, ischemic damage, infection,
neuroinflammation or neurodegeneration, and in addition to the
morphological changes, dramatic alterations in astrocyte gene
expression occurs (Hernandez et al., 2000; Pekny and Nilsson,
2005). The concept of reactive astrogliosis, especially its molecu-
lar and cellular characteristics, are still ill-defined, and we are only
starting to understand its multifaceted contribution to disease
pathogenesis and recovery from injuries (Barres, 2008; Burda and
Sofroniew, 2014; Zamanian et al., 2012). In reactive gliosis the
expression of many genes is altered, and these cells exhibit distinct
morphological and functional features. Given the molecular
complexity of the response of reactive astrocytes, it is unlikely that
a single molecular target or pathway will be implicated, indicating
that gliosis is a very complex reaction in which many elements are
implicated (Pekny and Pekna, 2014).
In response to infection and in certain inflammatory scenarios,
Müller cells undergo reactive gliosis. Müller gliosis is a generalized,
more unspecific, yet a very complex response of Müller glia towards
injury or infection. Müller cells become activated by nearly every
pathological challenge in the retina (Bringmann et al., 2009a).
20 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
Reactive gliosis is thought to be the result of signals received from
the injured or diseased tissue that sets off a molecular cascade
within the glial cells to provoke a change of state (Fischer and
Bongini, 2010). Typical features of Müller cell gliosis involve
cellular hypertrophy, up-regulation of IF expression (e.g., GFAP and
vimentin), increased rates of proliferation, loss of function,
inflammation, downregulation of GS expression, and tissue and
vasculature remodelling (Belecky-Adams et al., 2013).
A mild to severe continuum of the states of reactive gliosis may
exist. In the mild to moderate forms of gliosis, the cells may un-
dergo hypertrophy and show some changes to their functionality,
yet if the trigger is removed, the cells might revert back to their
former condition without altering the tissue. In the more severe
forms of reactive gliosis, cells undergo hypertrophy, lose their
functionality, form glial scars that are inhibitory to axonal regen-
eration and neuronal survival, and they may also proliferate The
more severe state is marked by the persistence of these charac-
teristics. Proliferative gliosis is generally associated with chronic
gliosis and it is usually detrimental but it can also have a beneficial
effect (Sofroniew, 2009). In such a state, the integrity of the
blooderetina barrier is lost and as a result, serum components leak
into the perivascular space, causing the re-entry of Müller cells into
the cell cycle and their proliferation (Bringmann et al., 2009a;
Coorey et al., 2012).
The release of VEGF by Müller cells under conditions stimulating
gliosis is especially interesting, as it acts in two directions. VEGF is
neuroprotective at low concentrations but at high concentrations it
causes all sorts of vascular pathologies. VEGF has neurotrophic ef-
fects (Zheng et al., 2012) and can prolong the survival time of cells
by inducing angiogenesis, which in turn attenuates brain edema
after cerebral ischemia/reperfusion and neuron injury. The neuro-
protective effect of VEGF was proposed to be unrelated to its
angiogenic effect but more closely related with its direct stimula-
tion of neuronal and endothelial activity (Oosthuyse et al., 2001).
The regeneration of neurons together with angiogenesis could be
improved by VEGF, especially after ischemia (Ma et al., 2011).
Conversely, the excessive and prolonged expression of VEGF has
devastating effects on the retinal vasculature and/or Müller cell
proliferation, which in turn might lead to retinal neuro-
degeneration (Tolentino et al., 2002). Moreover, calcium contrib-
utes to the high-glucose-induced expression of the angiogenic
factors HIF-1a and VEGF by retinal Müller cells. The activation of
the CaMKII-CREB pathway by high-glucose may be a possible
mechanism underlying the pathogenesis of diabetic retinopathy (Li
et al., 2012).
Müller cells are not alone in influencing the effects of VEGF as
astrocytes are also considered a target for ischemic damage. Apart
from their neuroprotective effect on neurons and their detoxifica-
tion of various ROS at the acute stage of ischemic stroke, reactive
astrocytes profoundly affect later post-ischemic stages. This influ-
ence may be mediated by the secretion of VEGF, which stimulates
the formation of new blood vessels and synaptogenesis, or by the
secretion of synaptogenic thrombospondin (Christopherson et al.,
2005). The positive stimulatory effect of VEGF on vessel and syn-
apse formation in ischemic stroke, contrasts with its induction of
blood-retinal barrier breakdown and lymphocyte infiltration in
autoimmune CNS inflammation (Argaw et al., 2009). It is also
noteworthy that treatment with GDNF enhances neuronal survival
in ischemic brain tissue and that GDNF is up regulated in reactive
astrocytes in the peri-infarct region (Kokaia et al., 1999). Netrin-4, is
a positive regulator of retinal angiogenesis and Müller cells
generate Netrin-4 in a hypoxia- and VEGF-dependent manner,
another example of a multifunctional protein produced by Müller
cells that may influence their complex relationship with retinal
neurons. Netrin-4 can support both axon guidance and
angiogenesis in early development, as well as modifying regener-
ative neurite growth and neovascularisation (NV) under patho-
logical conditions (Lange et al., 2012).
Surfactant protein A (SP-A) upregulates cytokine expression in
lung disease associated to prematurity. SP-A expression and local-
ization has now been characterized in the mouse retina, as has its
impact on NV in the mouse. Retinal and Müller cell SP-A is upre-
gulated via the NFkB pathway and it is up regulated during the
hypoxia phase of oxygen-induced retinopathy (OIR). A lack of SP-A
attenuates NV in the OIR model and thus, SP-A may be a marker of
retinal inflammation during NV (Bhatti et al., 2014). Müller cells
also produce PEDF (Pigment epithelium-derived factor), TGF-b and
thrombospondin-1 as anti-angiogenic proteins that are important
in adjusting the ‘‘angiogenic balance’’ (Eichler et al., 2004). Recent
studies suggest that in addition to PEDF (Yafai et al., 2007), TGF-b2
is one of the Müller cell derived factors that limit intra-retinal NV
(Yafai et al., 2014).
Within the mammalian retina, both the Müller glia and retinal
astrocytes react to injury and disease. Although the mechanisms
and triggers involved have not been fully characterized. A variety of
growth factor signalling mechanisms have been found to be
important in reactive gliosis, such as those of EGF, FGF, TNF-a,
ciliary neurotrophic factor (CNTF), insulin and WNTs (Nakazawa
et al., 2006; Sofroniew, 2009). Bone morphogenetic protein 7
(BMP7) also induces changes in the mRNA and protein of markers
typically associated with reactive gliosis in retinal astrocytes and
Müller glial cells, including GFAP, GS, a subset of CSPGs, MMPs, and
other molecules (Dharmarajan et al., 2013).
3.10. Neuroprotection
3.10.1. Neurotrophic factors
Neurotrophins and growth factors are involved in the survival,
differentiation and development of neurons. Administration of
different combinations of neurotrophins in the eye has been re-
ported to increase RGC survival in different experimental para-
digms. Glial cells are important to maintain the survival of neurons,
although the factors involved in the development and survival of
glial cells themselves are less well defined. Glial like Müller cells,
astrocytes and microglia are known to synthesise neurotrophic
factors and these can directly or indirectly mediate neurotrophic
actions (Garcia et al., 2003; Ruiz-Ederra et al., 2003; Vecino et al.,
1998a, 1998b). In order to define the cell-to-cell communication
between Müller glia and neurons, it is essential to first isolate the
respective cell types. When adult retinas were maintained in vitro
for 6 days, the expression of neurotrophins and their receptors was
preserved in glial cells and in RGCs, irrespective of the substrate on
which they were cultured (Fig. 10: Garcia et al., 2002). Moreover,
the factors secreted by Müller cells offered protection to RGCs
(Garcia et al., 2003).
The distribution of neurotrophins and their receptors in the
mammalian retina has been studied in detail, even in pathological
conditions like ischemia or following NMDA induced exitotoxicity
(Vecino et al., 1998b, 1999). BDNF expression by rat RGCs is
completely blocked after intravitreal injection of NMDA, as is
choline acetyltransferase (ChAT) immunoreactivity associated with
a subset of amacrine cells. However, immediately after NMDA in-
jection, BDNF mRNA expression by RGCs and its immunoreactivity
is enhanced, while the amacrine cell ChAT immunoreactivity is
clearly reduced, and expression of the mRNA transcripts encoding
rhodopsin and Thy-1 does not change. However, a few hours later
an increase in BDNF expression is no longer apparent, suggesting
that the synthesis of BDNF increases in RGCs immediately after an
NMDA insult a natural protective mechanism in retinal cells
(Vecino et al., 1999). A similar transitory increase of BDNF and its
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40 21

Fig. 10. Preservation of the neurotrophins and their receptors in (red) from adult pig retinal glia (in vivo: A, C and E) and primary cultured cells (in vitro: B, and F). (A and B) high-
affinity neurotrophin receptor, TrkA (red: C and D), low-affinity neurotrophin receptor, p75 (E and F) and neurotrophin-3 (NT3) (E). In D and F, retinal ganglion cells were stained
with anti-neurofilament antibodies (green). Scale bar ¼ 50 mm.

high-affinity TrkB receptor is also found in photoreceptors and
RGCs after ischemia-reperfusion. The mechanisms underlying
BDNF induced neuroprotection are unknown, although it has been
suggested that the BDNF derived upregulation of GLAST and GS
may contribute to this effect. Indeed, BDNF has a protective effect
on GLAST and GS expression by Müller cells under hypoxic condi-
tions, which could induce neuroprotection (Dai et al., 2012b).
The Müller cell extensions that form three laminae in the IPL of
the rat retina contain p75. After ischemia, there is a progressive
disorganization of the laminae that is worse after 24hr than after
2hr of reperfusion (Fig. 7). Müller cells modulate neuronal activity
by regulating the concentration of neuroactive substances and
potassium in the extracellular space by means of a variety of
transport processes (Newman and Reichenbach, 1996). Hence,
Müller cells are particularly important in ischemia when there is a
massive release of transmitters from neurons (Hossmann, 1993).
Thus, the changes seen in the p75 immunoreactivity associated
with the branches of the Müller cells in the inner plexiform layer
after ischemia may therefore indicate changes in plasticity and be
of functional significance (Vecino et al., 1998b). Disorganization of
22 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
Müller cell processes has also been observed in RP65 mutant dogs,
in which the primary cells affected are photoreceptors, while in
glaucomatous rat retinas the cells affected are RGCs. These changes
within the Müller cells again demonstrate that Müller glia have a
radial function in the retina that senses the changes in the outer as
well as the inner retina, reacting by modulating the structure and
possibly the function of retinal neurons.
3.10.2. Other trophic factors
Ciliary neurotrophic factor (CNTF) CNTF exerts numerous ef-
fects on the development, differentiation and survival of retinal
neurons (Rhee and Yang, 2010). The molecular mechanisms that
explain the neuroprotective role of CNTF in the retina include: a
direct action on photoreceptors to prevent apoptosis; the stimula-
tion of Müller cells to produce photoreceptor survival factors (Zack,
2000); enhanced synthesis or distribution of glutamate trans-
porters, thereby improving glutamate handling and resulting in
less excitotoxic damage to retinal neurons (Escartin et al., 2006);
and an induction of metabolic plasticity and increased resistance to
metabolic damage (Escartin et al., 2007). Defining the global tran-
scriptional response of Müller cells to CNTF in vivo will help
elucidate the molecular basis of its biological actions in the retina.
CNTF induces rapid and extensive transcriptional changes in Müller
cells and several genes induced by CNTF in normal Müller cells are
also up regulated in gliotic Müller cells involved in inherited and
experimentally-induced retinal degeneration. Hence, CNTF would
appear to be an inducer of gliosis in the retina. Interestingly, the
transcript profiles of Müller cells and astrocytes are similar
although they are derived from distinct cell lineages (Xue et al.,
2011). Exogenous CNTF initially targets Müller glia and they
trigger a reaction that induces the production of cytokines that act
through gp130 in photoreceptors to promote neuronal survival
(Rhee et al., 2013). Indeed, CNTF not only reduces the death of
photoreceptors but it also rescues retinal function (Heiduschka
et al., 2013). In addition, lens injury upregulates CNTF in retinal
astrocytes and Müller cells.
bFGF is a pleiotropic cytokine that, in addition to its pro-
angiogenic actions, may elicit further effects on retinal cells. bFGF
is found in astrocytes, Müller cells, RGCs and RPE cells. Although
bFGF is considered to be neuroprotective in the retina, it may also
have detrimental effects by stimulating aberrant vessel growth, or
inducing the proliferation and de-differentiation of Müller cells
(Bringmann et al., 2009a). Together, bFGF and VEGF act synergis-
tically on microvascular endothelial cells (Yan et al., 2001), whereby
the effects of bFGF are in part mediated by stimulating VEGF release
from Müller and vascular endothelial cells (Hollborn et al., 2004).
Müller cells represent a major source for bFGF in the ischemic
retina and in diabetic fibrovascular tissue. The secretion of bFGF by
Müller cells is stimulated by various pro-angiogenic growth factors
and cytokines. Indeed, while the soluble factors released by Müller
cells in response to transient hypoxia produce an anti-angiogenic
environment for retinal vascular endothelial cells, persistent hyp-
oxia increases bFGF release from Müller cells, which may become
increasingly important to shift the balance between stimulators
and inhibitors of angiogenesis in the retina. In such conditions
Müller cells are likely to provide a stimulatory environment for the
proliferation of retinal vascular endothelial cells and bFGF may play
a key role in the stimulation of abnormal vessel growth in the retina
(Yafai et al., 2013). In diabetes, the increased formation of advanced
glycation end-products (AGEs) in the vitreous humour may also be
involved in the initiation and progress of intraocular NV through
the production of bFGF by Müller cells (Ai et al., 2013).
PEDF (Pigment epithelium derived factor) Müller glial cells
are PEDF producers, a positive trophic factor expressed by activated
Müller cells. PEDF is a 50 kDa glycoprotein and it is upregulated by
Müller cells following retinal hypoxia or injury. PEDF functions as
an anti-oxidative and anti-inflammatory agent, as well as exerting
neuroprotective and neurotrophic effects that are mediated by
activation of the NF-kB (nuclear factor kappa B) and ERK1 and 2
pathways. When secreted by Müller cells, this factor acts directly on
PEDF receptors at the surface of RGCs. The upregulation of PEDF in
Müller cells occurs early in gliosis and it may reflect an attempt to
rescue retinal neurons from further damage (Unterlauft et al.,
2012). Indeed, PEDF reduces RGC loss in DBA/2J mice, a mouse
model of inherited glaucoma (Zhou et al., 2009).
The expression of PEDF, Kir4.1 and GLAST by retinal Müller cells
is unbalanced at high glucose concentrations, with abnormal
expression closely related to oxidative stress. Decreased expression
of PEDF induces a down-regulation of Kir4.1 in Müller cells, causing
membrane depolarization of Müller cells and GLAST dysfunction.
Thus, retinal PEDF must balance the anti-oxidative state against the
need to protect retinal neuronal and glial cells (Xie et al., 2012).
Further evidence for the neuroprotective actions of Müller cell
derived PEDF in the retina involves the intracellular signalling
molecules that may operate in the RGC under conditions in which
PEDF of glial origin exerts its pro-survival effects on RGCs. Thus,
PEDF-induced NF-kB activation enables RGCs to protect themselves
from hypoxia- or growth-factor deprivation-induced apoptosis.
Although not directly demonstrated, exposing RGCs to PEDF may
increase the phosphorylation of the inhibitor NF-kB, I-kB, thereby
unmasking the NF-kB/NLS domain at the C terminus of NF-kB,
stimulating its nuclear translocation and increased its DNA bind-
ing activity (Unterlauft et al., 2014).
Insulin-like growth factor-1 (IGF-1) is a polypeptide growth
factor similar in structure and function to insulin. IGF-1 has mito-
genic, metabolic and growth-stimulating activities in many cells
and tissues, including the retina, and it promotes the survival of
various cells following serum deprivation. This protective effect of
IGF-1 most probably occurs via PI3K/Akt, although the complete
details of the upstream and downstream targets have not yet been
elucidated (Guvakova, 2007).
Glial Derived Neurotrophic Factor (GDNF) in the retina has
been reported to rescue photoreceptor activity in the rd1 mouse
model of Retinitis Pigmentosa (Frasson et al., 1999) and its effects
are transmitted indirectly via activation of Müller glia (Harada et al.,
2003; Hauck et al., 2006). Transcriptome profiling of the mouse
retina in response to GDNF identified osteopontin (OPN) as a novel
GDNF effector that could in part mediate in the GDNF induced
neuroprotective activity of Müller cells on photoreceptors (Del Rio
et al., 2011). OPN is secreted by Müller glia and it supports photo-
receptor survival in explant cultures of rd1 retinas, potentially
exerting a direct pro-survival effect on photoreceptors. OPN has a
significant, concentration-dependent survival effect on primary
porcine photoreceptor cells, and it activates the PI3K/Akt pro-
survival pathway (Del Rio et al., 2011). OPN also induces the
release of VEGF, glutamate, ATP and adenosine from Müller cells.
The effect of OPN was prevented by blockers of voltage-gated so-
dium channels (tetrodotoxin), T-type voltage-gated calcium chan-
nels (kurtoxin), potassium channels (clofilium) and chloride
channels (5-nitro-2-(3-phenylpropylamino)-benzoic acid) (NPPB).
The swelling-inhibitory effect of OPN was dependent on intracel-
lular calcium signalling, the activation of PLC and protein kinase C,
and vesicular exocytosis of glutamate. In retinal slices, Müller glial
cells are immunoreactive for OPN and Müller cell-derived OPN has
autocrine effects. The neuroprotective effects of OPN may in part be
mediated by preventing the cytotoxic Müller cell swelling, and the
release of VEGF and adenosine by Müller cells (Wahl et al., 2013).
VEGF (see section on Gliosis above).
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
3.11. Phagocytosis
The phagocytotic activity of Müller cells was described more
than eight decades ago. There is no evidence that these cells can
phagocytise pathogens, however the phagocytotic properties of
Müller cells were recently revaluated following Staphylococcus
aureus infection. As a result, Müller cells can apparently not only
phagocytise bacteria but they can also kill them intracellularly
(Singh et al., 2014).
Astrocytes play a role in the life cycle of axonal mitochondria at
the head of the optic nerve. Optic nerve fibres discard their mito-
chondria, which are then engulfed by neighbouring astrocytes
(transmitophagy: Davis et al., 2014). In the myelination transition
zone, astrocytes have a constitutive phagocytotic role and they
express the phagocytotic marker Mac-2, which augments in glau-
coma, and it may play an important role in axonal degeneration
(Nguyen et al., 2011).
The death of RGCs after optic nerve injury involves the partici-
pation of microglia/macrophages and astroglia in a neuro-
inflammatory scenario. Simvastatin, a statin used to lower blood
cholesterol, may reduce NF-kB activation by TNF-a in astrocytes
maintained in culture. When administered systemically, the drug
suppresses RGC death possibly by ameliorating the astroglial re-
action to injury (Morishita et al., 2014). TNF-a can activate signal-
ling reactions involving NF-kB or C-Jun N-terminal kinase (JNK)
pathways. These pathways influence neurodegeneration and serve
as neuroprotectors. The detrimental and neuroprotective role of
TNF-a signalling was recently assessed in cultured RGCs and as-
trocytes (Dvoriantchikova and Ivanov, 2014), apparently activating
NF-kB and JNK in astrocytes but not in RGCs, indicating a contrast in
both cell populations. However, the significance of this finding
in vivo remains unclear. TNF-a also induces the secretion of MMP-2
by optic nerve astrocytes and this secretion is attenuated by a se-
lective delta opioid agonist, SNC121 (Akhter et al., 2013).
Extracellular ATP acts as a ligand to purinergic receptors and
P2X4 purinergic receptors (ionotropic receptors) are distributed in
different cell types within the retina (bipolar cell terminals, hori-
zontal cell somata and processes, calretinin-positive amacrine
cells…etc), microglia, Müller cells and astrocytes. These receptors
influence glia function and local tissue homeostasis in both physi-
ological and pathological conditions (Vessey et al., 2014).
3.12. Repair and regeneration
Retinal astrocytes and Müller cells both produce multiple factors
that are trophic to RGCs, perhaps at different levels and under
different conditions. RGCs express a number of receptors that
respond to trophic factors including but not restricted to leukaemia
inhibitory factor (LIF), CNTF, NGF, BDNF, IGF, neurotrophins, GDNF
and FGF2 (Bonnet et al., 2004; Kinkl et al., 2003; Vecino et al.,
1998b, 2002). However, promotion of RGC survival is only tran-
sient and does not necessarily correspond with the restoration of
axonal outgrowth. In fact, whilst some of these trophic factors are
very efficient at enhancing cell survival, some of them promote
axonal growth (Vecino et al., 2002), indicating that a delicate bal-
ance must exist to promote optimal RGC regeneration.
Reactive glia clearly display inhibitory properties, yet an inter-
mediate “activated” glial phenotype may also exist that favours
axonal regeneration by upregulating factors that enhance their
growth and repair, whilst keeping inhibitory responses below the
threshold for damage. Acute activation of glia is neuroprotective,
while sustained activation is detrimental, for example due to the
release of inflammatory cytokines. Enhanced reactivity or “activa-
tion” of retinal glia has positive effects on RGCs due to the pro-
duction of trophic factors, both in vivo after injury and also in vitro.
23
Indeed, reactive glia secrete trophic factors distinct to those pro-
duced by quiescent glia and the degree of gliosis also appears to be
crucial to determine the success of RGC regeneration.
Can retinal glia be primed to have positive or negative effects on
RGC survival and regeneration? Müller cells secrete factors to the
media that favour the survival, regeneration and branching of RGC
axons in vitro (Garcia and Vecino, 2003; Higginson et al., 2013).
Activation of rat retinal glia (astrocytes and Müller cells), either by
lens lesion, intravitreal peripheral nerve grafting, zymosan injec-
tion or experimental glaucoma (Lorber et al., 2009, 2008, 2002,
2012), enhances the regenerative potential of RGCs in vivo and
in vitro. It is assumed that retinal glia are activated by factors pro-
duced after lens injury or peripheral nerve graft, or by macro-
phages, which may be proteinaceous or non-proteinaceous, and
that they are then stimulated to secrete neurotrophic factors that
enhance the growth of RGC axons. A trophic factor produced by
activated astrocytes and Müller cells that enhances RGC axonal
regeneration is CNTF. Its production is mediated by apolipoprotein
E (ApoE), which is upregulated in retinal astrocytes and Müller cells
after optic nerve crush and intravitreal Zymosan injections (Lorber
et al., 2009; Muller et al., 2007). Other trophic factors produced by
reactive retinal astrocytes and Müller cells that may be involved in
RGC regeneration include BDNF, GDNF and bFGF (Bonnet et al.,
2004; Garcia et al., 2002, 2003; Garcia and Vecino, 2003; Harada
et al., 2000, 2002Kinkl et al., 2003).
Although mammalian Müller glia can respond to injury, prolif-
erate and express genes that are associated with retinal stem cells
(Roesch et al., 2008), they do not function as retinal progenitors
in vivo. Nonetheless, these characteristics suggest that under the
right circumstances, Müller glia might act as retinal progenitors
that can be used for repair. The regenerative potential of Müller
cells makes them ideal candidates for cell replacement therapy to
treat retinal degenerative diseases, such as age-related macular
degeneration, retinitis pigmentosa or glaucoma, where loss of
vision correspond to retinal neuron loss. A regenerative strategy
that mobilizes the patients’ own Müller progenitors to participate
in retinal repair would have several advantages over cell trans-
plantation therapy (Ruilin et al., 2014). Indeed, in rodent and hu-
man cell cultures, Müller glia can generate both neurons and glia
(Das et al., 2006). Importantly, primary human Müller glial cell
cultures can generate photoreceptors and RGCs that have some
reparative potential when transplanted into a damaged rodent
retina. These studies suggest that human Müller glia are capable of
generating neurons under appropriate conditions and that they
may be able to participate in repair. Indeed, human Müller glia stem
cells (hMGSCs) can differentiate into functional RGC precursors
in vitro, as confirmed by their expression of RGC markers and the
increased cytosolic calcium in response to nicotinic stimulation
(Singhal et al., 2012).
Viral expression of ASCL1 is sufficient to activate a neurogenic
program in mammalian Müller glial, both in dissociated cultures
and in the intact retina. ASCL1 remodels the chromatin of retinal
progenitor genes, activating their expression and downregulating
glial genes. The reprogrammed Müller glial differentiate into cells
that resemble neurons morphologically, both in terms of gene
expression and in their responses to neurotransmitters. These re-
sults suggest that stimulating neurogenesis in Müller glial with
ASCL1 could provide an alternative strategy for retinal repair after
disease or injury (Pollak et al., 2013). Similarly, manipulating the
Ephrin-A2/A3 pathway may represent a viable approach to pro-
mote the progenitor cell fate of Müller cells, since Ephrin-A2 and A3
inhibit the regenerative potential of Müller cells in the retina of
adult mice (Ruilin et al., 2014).
Among the possible molecular and cellular mechanisms con-
trolling neuronal regeneration, p53-mediated cell cycle arrest may
24 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
play an important role in preventing Müller glia proliferation in the
adult mouse retina (Ueki et al., 2012). The loss of p53 activity could
significantly enhance the proliferation potential of cells and facili-
tate the reprogramming of Müller glia into retinal progenitor cells
(Zhao et al., 2014a). The tumour suppressor p53 plays a funda-
mental role in astrocytes, when environmental conditions are
deleterious, as in heightened conditions of oxidative stress. In
“super p53” mice that carry two copies of the p53 gene, the density
of astrocytes is greater than in the wild type and the area they
occupy is also augmented (Salazar et al., 2013).
Atoh7 is an essential transcription factor that determines the
competence state of RGC progenitors. Ectopic expression of Atoh7
significantly increases the number of RGCs that differentiate from
cultured retinal stem cells in vitro (Yao et al., 2007). Moreover,
Atoh7 promotes the differentiation of Müller cell-derived retinal
stem cells into RGCs by inhibiting Notch signalling, opening up a
new avenue for gene therapy and optic nerve regeneration in
glaucoma (Song et al., 2013).
Glutamate is the main neurotransmitter of the vertical signal-
ling pathway in the retina and it induces the expression of pro-
genitor cell markers in adult murine Müller glia, both in vivo and
in vitro (Takeda et al., 2008), as well as promoting the proliferation
of Müller derived progenitor cells (Ramirez and Lamas, 2009).
Glutamate can trigger de-differentiation in primary cultures of rat
Müller glial cells through a process that involves the activation of
NMDA and group II metabotropic glutamate receptors, and to some
extent the participation of global DNA demethylation via gadd45b
(Reyes-Aguirre et al., 2013).
4. Specific functions
4.1. Light guidance of Müller cells
In the mammalian retina, light has to pass the entire depth
before it arrives at the photoreceptors suggesting that specific
retinal tissue optics overcome light scattering, even in the typical
non-foveate vertebrate retina. It has been proposed that Müller
radial glial cells might act as light-guiding elements (Labin and
Ribak, 2010) and that to minimize light scattering, non-foveal
Müller cells act as living optical fibres that guide light through
the inner retinal layers towards the photoreceptors, similar to glass
fibre optics (Franze et al., 2007). The funnel-shaped endfoot of
Müller cells could serve as a light collector at the vitreal surface of
the retina and the stem processes as light-guiding fibres. Between
the vitreous and the retina there is a ‘soft coupling’ of the light path
that reduces light reflection at the inner retinal surface (Franze
et al., 2007). Müller cells in the living retina minimize intraretinal
light scattering, conserving the diameter of the beam that hits a
single Müller cell endfoot. Thus, high intensity light reaches the
individual photoreceptors. By minimizing scattering and enhancing
the signal/noise ratio, Müller cells conserve the spatial distribution
of light patterns in the propagating image. The ratio between the
numbers of cones and Müller cells is roughly 1:1, indicating an
optimal coupling between the light-guiding units and the func-
tional light pattern-sensing units (the cones: Agte et al., 2011). The
molecular basis of the light-guiding capacity of Müller cells remains
to be determined, although bundles of IFs are arranged along the
light path. In tissue edema, the neuroretina loses its transparency
and the milky opacity of the edematous tissue makes it likely that
the light guidance through Müller cells is deteriorated, resulting in
increased scattering (Reichenbach and Bringmann, 2013).
4.2. Progenitor cells
Unlike neurons, glia have the lifelong capability to de-
differentiate and re-enter the mitotic cycle. Under pathological
conditions, hypertrophy and the proliferation of glial cells leads to
the formation of glial scars, which fill the spaces left by dying
neurons and disconnected synapses (Reichenbach and Bringmann,
2010). Müller glia in mammalian retinas can respond to injury,
proliferate and express genes that are associated with retinal stem
cells (Roesch et al., 2008), displaying a gene expression profile
resembling that of progenitor cells (Blackshaw et al., 2004). A
subset of Müller cells express progenitor genes like Car2, Dkk3,
Chx10 and Pax6 (Roesch et al., 2008; Rowan and Cepko, 2004), and
Müller cells in the adult mouse retina express cell cycle genes like
cyclinD3, Cdc14A, Cdk10 and Spbc25, as well as genes in the Notch
pathway that might regulate the re-entry of Müller glia into the cell
cycle under pathological conditions (Roesch et al., 2008).
Müller glia might adopt characteristics of retinal progenitors
that can be used to repair injured retina. In chicks (Fischer and Reh,
2001) and rats (Das et al., 2006; Ooto et al., 2004) Müller cells may
even differentiate into cone and rod photoreceptors, raising the
possibility that they may play a role as retinal stem cells. Indeed,
primary human Müller glial cell cultures can generate photore-
ceptors and RGCs that have some reparative potential when
transplanted into a damaged rodent retina (Jayaram et al., 2014).
Thus, Müller glia might be the ideal candidate for cell replacement
therapy to treat retinal degenerative diseases where vision loss is
caused by retinal neuron loss (Ruilin et al., 2014).
Müller glia with stem cell characteristics have been identified in
the adult human eye and although there is no evidence that they
regenerate the retina in vivo, they can be isolated and grown in
culture and can be induced to express retinal neuron markers
in vitro (Lawrence et al., 2007). Both hMGSCs and RGC precursors
differentiate, as shown by the expression of RGC markers and the
increase in cytosolic Ca2þ in response to nicotinic stimulation
(Singhal et al., 2012), the former generating functional newly born
RGCs in response to BMP, FGF2 and the secretase inhibitor (DAPT).
This provides important evidence that hMGSCs can be differenti-
ated in vitro into functional RGCs, providing a potential application
in cell-based therapies to treat conditions in which RGCs are
compromised (Becker et al., 2013). In addition, an immortalized
human Müller glial cell line (MIO-M1) has been established that
can maintain its characteristics over 50 sub-culture passages (Limb
et al., 2002).
Cell-derived micro vesicles (MVs: recognized as important
components of cellecell communication) contain mRNAs, miRNAs,
proteins and lipids, and they can transfer their bioactive contents
from parent cells to cells of other origins. Embryonic stem cell
microvesicles (ESMVs) induce de-differentiation and the pluripo-
tency of Müller cells after prolonged exposure, opening an avenue
to employ ESMVs in therapies to induce and support the retina's
endogenous regenerative capacity (Katsman et al., 2012).
The viral expression of ASCL1 is sufficient to activate a neuro-
genic programme in mammalian Müller glia, both in dissociated
cultures and in the intact retina. The reprogrammed Müller glia
differentiate into cells that morphologically resemble neurons, in
both their gene expression and their responses to neurotransmit-
ters. Hence, stimulating neurogenesis in Müller glia with ASCL1
could provide an alternative strategy for retinal repair after disease
or injury (Pollak et al., 2013). Manipulating the ephrin-A2/A3
pathway may also represent an alternative means to promote the
progenitor cell fate of Müller cells (see above: Ruilin et al., 2014).
Among the possible molecular and cellular mechanisms that
control neuronal regeneration, p53-mediated cell cycle arrest may
play an important inhibitory role in Müller glia behaviour in the
adult mouse retina (Ueki et al., 2012). Loss of p53 activity can
significantly enhance the proliferation potential of cells and facili-
tate the reprogramming of Müller glia into retinal progenitor cells
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
(Zhao et al., 2014a). The tumour suppressor p53 may also strongly
influence astrocytes (see above). The possible influence of Atoh7
and glutamate in promoting the generation and proliferation of
Müller glia derived progenitor cells has also been addressed above.
Little is known about the role of BMPs in Müller glial prolifer-
ation although a novel role for BMP in the control of Müller glial
proliferation was recently described. Treating the mouse retina
with EGF stimulates the BMP signalling pathway in Müller glia,
causing them to release BMP7. This autocrine BMP release activates
the BMP receptors on Müller glia, promoting Smad1/5/8 phos-
phorylation and an increase in Id1. Hence, BMP can influence the
mitogenic pathway regulating Müller glial proliferation, which has
implications for both vitreoretinopathy and regenerative ap-
proaches to address retinal disease (Ueki and Reh, 2013).
The capacity of some astrocytes to enter a re-programmed state
of pluripotency and subsequently, to serve as progenitors capable of
generating neurons suggests a reparative function that merits
attention (Buffo et al., 2008; Wang et al., 2008; Wohl et al., 2012) In
fact, the generation of neurons from astrocytes has been reported
in vitro and it depends on specific transduction mechanisms
mediated by key transcription factors like Pax 6 (Heins et al., 2002)
or Mash 1 (Ding et al., 2014).
5. Heterogeneity of retinal glial cells
5.1. Astrocyte heterogeneity
The phenotypic heterogeneity among glial cell types is an
emerging area of research in glial biology. Whilst most of the
attention to date has focused on astrocyte subpopulations in the
brain white matter (Chaboub and Deneen, 2012), there is some
evidence to support the existence of glial subpopulations within
the adult mammalian retina. At least nine types of astrocytes with
different morphological characteristics have been described in the
normal brain. Moreover, morphologically undistinguishable astro-
cytes can exhibit different physiological behaviour (Matyash and
Kettenmann, 2010). The existence of different astrocyte pop-
ulations is reinforced by the finding that astrocyte reactivity takes
place in selected areas of the glaucomatous retina, these “micro-
domains” possibly serving as biomarkers and predictors of RGC
health (Formichella et al., 2014).
Astrocytes form a family of diverse populations adapted to their
specific localization. Astrocytes in different brain regions have
distinct physiological properties, and they express various sets of
receptors and transporters, probably in response to their local
environment. Within the retina the situation is similar and when
primary retinal astrocytes are cultured, different types are evident
that vary in their expression of neurotransmitters and molecular
Fig. 11. Astrocyte heterogeneity. (A) Pig retinal astrocytes labelled with antibodies against GFAP. (B) Astrocytes labelled with fluoro-gold FG injected into the optic nerve blood
vessels of a pig eye ball that was preserved in vitro for 12 h in oxygenated medium. Note that in A there is at least one other type of astrocyte not associated with the blood vessels
and located in parallel to the RGC axons.
25
markers, and in their shape. For example, AQP4 is present in large
amounts in the astrocyte plasma membranes when they are in
contact with microvessels. AQP4 influences many process (the
volume of the extracellular space, waste clearance, potassium
buffering, cell migration and calcium signalling: Nagelhus and
Ottersen, 2013) and significantly, its expression is altered in dia-
betic retinopathy (Qin et al., 2012). Astrocytes may play a role in the
transport of exosomes with different cargos between neurons
(Edelstein and Smythies, 2014; Kawikova and Askenase, 2014). In a
laser-induced choroidal neovascularisation (CNV) model, retinal
astrocytes release exosomes containing anti-angiogenic com-
pounds that protect the eye from the deleterious effects of neo-
vascularisation (Hajrasouliha et al., 2013).
Differential expression of TrkA, the NGF receptor, led us to
identify two sub-populations of astrocytes in the adult human and
pig retina. While most astrocytes in the nerve fibre layer (NFL)
express TrkA, a small population in the INL do not express this
receptor (Ruiz-Ederra et al., 2003). A separate study of adult human
retinal astrocytes identified 3 different sub-populations character-
ized by the expression of GFAP and Vimentin; GFAPþ/Vimentinþ,
GFAPþ/Vimentin and GFAP/Vimentinþ (Perez-Alvarez et al.,
2008). The physiological significance of this, if any, is not yet
known but these data provide further strong evidence for the
heterogeneity of retinal astrocytes.
In addition to the two sub-populations of astrocytes showing
distinct TrkA expression in the INL, we found another possible sub-
population of retinal astrocytes based on their function. Thus, only
a subpopulation of astrocytes were able to take up Fluorogold
injected systemically in the blood vessels of the optic nerve of pig
eyes when incubated over night in oxygenated cultured medium.
Given their morphology, we propose that these specialized cells are
the functional “perivascular astrocytes” described previously in the
human eye (Wolter, 1956). These astrocytes are preferentially
connected to blood vessels while the rest of the astrocytes in the
NFL of the retina connect mainly with RGC axons (Fig. 11).
Interspecies differences have been observed in the distribution
of astrocytes, which is very similar in the pig and human where two
types of astrocytes are found: stellate astrocytes that form cell
clusters; and elongated astrocytes located in the NFL (Ruzafa et al.,
2014). By contrast, only stellate astrocytes are found in rodents.
Interspecies differences in brain astrocytes have also been found
and differences exist between rodent and human astrocytes in
similar areas of the cortex, including differences in size, branching
area and calcium waves (Oberheim et al., 2009).
5.2. Müller cell heterogeneity
Little is known about the heterogeneity of Müller cells, although
26 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40

only 30% of the Müller cells in vitro express class II MHC antigen,
suggesting that these might be involved in retinal immune re-
actions (Roberge et al., 1985). In an attempt to define the tran-
scriptome of Müller glia, certain genes were seen to be expressed
heterogeneously among the Müller glial cells including common
housekeeping genes like b-actin, b-2-microglobulin, heat shock
protein 1 b and TATA box binding protein (Roesch et al., 2008). It is
clear that molecular differences exist in Müller cells because the
homeodomain containing transcription factor Chx10 is expressed
only in a subset of Müller glia (Rowan and Cepko, 2004). Hetero-
geneous gene expression among Müller glial cells was observed
over a large cluster, yet further characterization of the glial genes
identified will be required to clarify the diversity among Müller
glial cells and to shed light on their potential functional diversity
(Roesch et al., 2008). A comparison of the Müller glia transcriptome
with that of other glial cell types in the CNS failed to reveal common
clusters of gene expression (Bachoo et al., 2004). This might be due
to technical differences or it might reflect a distinct genetic
constitution. However, the expression of some transcripts was
common to Müller glia and other glial cell types, and some tran-
scripts enriched in Müller glia are also enriched in astrocytes
include peroxiredoxin 6, Basigin (also called CD147), caveolin, CD9
antigen, carboxypeptidase (Roesch et al., 2008).
Isolated cells in vitro can respond to certain conditions by
rapidly changing their protein expression and transforming to a
proliferative phenotype. While vimentin remains stably expressed
in these cells, GFAP expression, which is probably induced during
their isolation from the intact retina, is rapidly lost in vitro (Fischer
et al., 2004). This is consistent with reports from the chick retina
Fig. 12. Müller cell heterogeneity in vitro. Confluent pig Müller cells monolayer
where only the sub-population of GFAP negative retinal Müller glia
cultured for 10 days and labelled with (A) antibodies against-GFAP (green) and (B)
become proliferative following NMDA-induced retinal damage, glutamine synthetase (GS, red). C) Co-localisation of both antibodies plus nuclei
possibly indicating that the primary retinal Müller glia represent stained with DAPI (blue). Note that even when there are many Müller cells in a field,
only a glial subpopulation (Hauck et al., 2014). there are only two cells with strong GS-immunofluorescence: one with strong GFAP
Other evidence for phenotypic diversity among Müller glia in- fluorescence and another with an intermediate strength of GS fluorescence but
negative for GFAP (top right). Scale bar ¼ 50 mm.
cludes the variation in nestin expression in Müller cells after retinal
detachment from adult rats (Luna et al., 2010). A different post-
injury response may be elicited in individual Müller cell pop- which all cells in a tissue participate. Likewise, when things go
ulations and since nestin is a marker of immature Müller cells, wrong due to trauma or disease, the community of retinal cells
perhaps sub-populations exist with enhanced regenerative prop- responds and collaborates to restore a healthy state. However, this
erties. Furthermore, a gene expression analysis of Müller cells in a cannot always be achieved and the damage may seriously and
mouse model of retinitis pigmentosa revealed significant inherent irreversibly affect retinal function. Indeed, many pathological
heterogeneity amongst individual wild type cells, with further di- conditions may alter the integrity of the retina. In the case of
versity amongst individual cells post-injury (Roesch et al., 2012). neurodegenerative diseases the role of microglia is variable, and
Heterogeneity in GS or GFAP expression was observed in pri- both their pathogenic engagement and neuroprotective role are
mary Müller cell cultures from adult rat or pig, and cells expressing entangled (i.e.: capacities and limits). Thus, both these roles must
high amounts of GS or GFAP lie close to others that do not express be fully understood to adequately design external interventions
these markers (Fig. 12). This was evident in both pure glial cell aimed at preventing, alleviating or eliminating the damage (Prinz
cultures as well as in Müller-RGC co-cultures. In addition, RGCs et al., 2011).
prefer to grow on Müller cells that overexpress GS, their regener- Retinal damage results from the action of different etiological
ating axons following a very precise path over the surface of Müller agents, such as elevated intraocular pressure, altered vascularisa-
cells that contain GS and avoiding the surrounding cells without GS tion, ageing, genetic defects. In different retinal neurodegenerative
(Fig. 13). This heterogeneity among Müller cells would perhaps also diseases the cell signals and changes, like the inflammatory re-
reflect different glialeneuronal interactions that may have neuro- sponses, exhibit both similar and singular characteristics, and
protective effects on retinal neurons. therefore, therapeutic interventions must be based on knowledge
Reactive Müller cells upregulate GS, possibly as a neuro- of the common and distinct patterns of the disease (Cuenca et al.,
protective mechanism to limit glutamate neurotoxicity (Gorovits 2014). The effect of a glial response contralateral to the damaged
et al., 1997). Whilst this is not a direct interaction between glia eye has been demonstrated and it is very possible that inflamma-
and RGCs, it does represent an important effect of glia on RGC tion caused by the way to increase intraocular pressure with laser
survival and retinal homeostasis. could be the cause of such contralateral reactions (Gallego et al.,
2012; Ramirez et al., 2010; Rojas et al., 2014). It is thus very
6. Glia and retinal pathologies important to pay attention to the inflammation caused in the
different experimental models of eye damage since in addition to
Microglia perform valuable activities in the retina under phys- elevation of intraocular pressure, it is possible that other retina
iological conditions by interacting with other cell populations in damage can be caused to the retina.
the retinal layers. Maintenance of homeostasis is a complex task in
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
Fig. 13. Preference of RGCs grown in vitro on Müller cells that express GS strongly. (A)
Rat RGCs were cultured on confluent pig retinal Müller cells monolayer where the
nuclei are labelled with DAPI (blue), the glutamine synthetase is labelled in Müller
cells (GS, green) and the RGCs are stained with antibodies against-ßIII Tubulin (red).
(B) RGCs have a preference for those Müller cells that express GS strongly. Scale
bar ¼ 50 mm.
6.1. Diabetic retinopathy
Diabetic retinopathy (DR) is a long term complication of type 1
and type 2 diabetes. In both the proliferative (angiogenesis
observed) and non-proliferative (disrupted blood vessels detected)
forms of DR, activation of a plethora of signalling pathways ulti-
mately results in retinal damage and visual loss (Grigsby et al.,
2014). Dysregulated metabolites (including glucose, amino acids,
lipids, amines, vitamins, minerals, etc) disturb the homeostasis of
growth factors, neurotrophic factors, cytokines and adhesion mol-
ecules, consequently affecting capillary permeability and cell
turnover. Therefore, many related and unrelated biochemical
pathways are involved in the complex pathophysiology of DR (Ola
et al., 2012), which explains why a precise picture of the mecha-
nisms related to the onset and progression of the disease is not yet
clear. Moreover, individual genetic susceptibility and habits influ-
ence the development and morbidity of the disease, which may
eventually lead to partial or total loss of vision as a consequence of
retinal destruction and/or detachment (Grigsby et al., 2014).
Clearly, DR has an important inflammatory component, where
microglia participate as both prey and enforcer, together with other
27
retinal cell populations, including neurons, glial cells and reduced
myeloid populations (for a review see (Grigsby et al., 2014).
As a consequence of consistent high blood glucose levels in DR,
AGE appear (molecules that are non-enzymatically glycated in the
presence of aldoses), dyslipemia occurs and there is an increase in
ROS that activates retinal microglia. As a result, the microvascula-
ture, as well as other glia and neurons suffer damage (Fletcher et al.,
2005; Grigsby et al., 2014; Wong et al., 2011). Various experimental
animal models of DR have provided useful information about the
molecular events involved in the disease. Alloxan or STZ (strepto-
zotozin) are toxic agents that specifically destroy pancreatic islet
beta cells and induce diabetes (Larger et al., 1995; Mansford and
Opie, 1968), while Goto Kakizaki (GK) rats are a non-obese model
for spontaneous type 2 diabetes developed by select breeding
(Kitahara et al., 1978). The retina of these experimental animals can
be used to analyse the role of microglia, in vivo and in vitro: their
migratory, secretory and growth activities, as well as their neuro-
protective capacity. Also, human subjects can be used to study the
disease, although with evident limitations.
Microglial activation in DR has been observed at early stages of
this progressive disease and an increase in microglial density oc-
curs in the GCL and NFL, with a decrease in the IPL. The alignment of
microglial processes along the optic nerve is also disrupted (Chen
et al., 2014). It is noteworthy that the precise mechanism(s) of
glial activation in DR and other retinopathies are not fully under-
stood. A few examples illustrate the specific and partial relevance of
several actors. The accumulation of AGA (Amadori-glycated albu-
min), an AGE product in early DR induces TNF-a secretion by
microglia (Ibrahim et al., 2011). Moreover, in an experimental rat
model of diabetes, activated microglia more intensely express TNF-
a and IL-1b mRNA, and interestingly, minocycline (a tetracycline
antibiotic with anti-inflammatory activity) produces an approxi-
mate 50% decrease in the transcripts encoding these two cytokines
(Krady et al., 2005). Another agent analyzed in DR is aldose
reductase (an enzyme that catalyzes the synthesis of polyols),
which is activated in DR (Kinoshita, 1986). Aldose reductase par-
ticipates in the activation of retinal microglia and its inhibition or
its genetic deficiency significantly reduces the inflammatory
changes caused by microglia activation (Chang et al., 2014),
ameliorating the degeneration of capillaries and the generation of
superoxide anions found in DR (Tang et al., 2013). The participation
of signalling pathways related to the microglial adenosine receptor
A2A has also been documented (see Santiago et al., 2014).
In cases of disease or trauma, astrocytes react and become
activated (Pekny et al., 2014; Yang et al., 2014), undergoing a series
of changes (proliferation/migration, hypertrophy, GFAP expression,
secretion of pro-inflammatory signals that include IL-6, MCP-1 and
MIP-2alp) aimed at tackling the insult in cooperation with other
glial cells (Nahirnyj et al., 2013). As a consequence of astrocyte
activation, RGCs receive both positive and negative signals, and the
disturbance of the homeostatic scenario triggers the death of
resident retinal cells. Astrocytes also respond to aggression and in
diabetes for example, high glucose levels alter astrocyte inflam-
matory cytokine expression, activating NF-kB and increasing
oxidative stress (Shin et al., 2014). In glaucoma and other degen-
erative retinal diseases, oxidative stress and death by apoptosis are
known features, as are astrocytic TNF-a/TNFR signalling, nuclear
factor (NF-kB) activation, and p53 and caspase 3 upregulation
(Rogers et al., 2012; Tezel et al., 2012). In neurodegeneration glial
activity also deteriorates.
Elevation of histone acetylation in Müller cells plays an impor-
tant regulatory role in the inflammatory response during diabetes
(Wang et al., 2012). It has also been proposed that in early phases of
diabetic retinopathy, Müller glial activation does not contribute to
neurodegeneration but that it may provoke neuroprotection
28 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
against high glucose induced neurotoxicity through a mechanism
involving phosphorylation of extracellular signal-regulated kinase
1/2, pERK1/2: (Matteucci et al., 2014). Since in vitro high glucose
stimulation of Müller cells increases VEGF secretion, the specific
ERK1/2 inhibitor, U0126, blocked ERK1/2 phosphorylation and
thus, impaired the Müller cells secretion of VEGF that is implicated
in the development of diabetic retinopathy (Ye et al., 2010). Thus,
targeting ERK1/2 signalling in Müller cells, through the upstream
signalling pathway or nuclear transcription factors of VEGF secre-
tion, could represent a promising future treatment for diabetic
retinopathy.
6.2. Glaucoma
Glaucoma encompasses several diseases that destroy RGCs and
their axons in the optic nerve through apoptosis (Garcia-Valenzuela
et al., 1995), provoking progressive loss of the visual field and
blindness (Mi et al., 2014; Quigley, 2011; Rieck, 2013). The aetiology
and pathophysiology of glaucoma is not well known, although
several risk factors and mechanisms that influence the disease have
been found. Among these factors, the elevation of intraocular
pressure (IOP) is particularly important, while other factors have
also all been associated with the disease, like: low diastolic ocular
perfusion pressure (calculated as 2/3 mean arterial pressure-IOP),
other ocular hemodynamic parameters, systemic diseases such as
sustained high blood pressure or diabetes, ethnic group, age,
myopia, migraine, the nutritional state of RGCs (Hernandez et al.,
2009).
Human glaucoma can be classified into three main categories:
primary open angle glaucoma (POAG), primary closed-angle glau-
coma (PCAG) and primary congenital glaucoma (PCG: Ahonen et al.,
2014; Quigley, 2011). Each of these has specific and common
characteristics, related to alterations of the aqueous humour
outflow and abnormalities in the anterior chamber angle (Ahonen
et al., 2014; Liu et al., 2013). The structural changes in the outflow
system involve a thickening of the trabecular beam, cell death in
the cribriform layer, accumulation of extracellular material and
plaque formation (Fuchshofer et al., 2006; Rohen et al., 1981).
However, a clear relationship between elevated IOP, structural al-
terations to the Schlemm's canal cells that respond to local me-
chanical changes, and the preponderant intervention of the
mechanisms related to the onset and progression of the disease are
not yet fully known (Galdos and Vecino, 2011; Overby et al., 2014;
Vecino et al., 2015a) Interestingly, therapies that lower IOP slow the
progress of different types of glaucoma, yet other treatments aimed
at avoiding cell death are required.
Due to the limitations faced when studying human individuals,
animal models of induced or spontaneous glaucoma are efficient
tools to discover and define the aetiological factors, pathological
mechanisms, and the extent and consequences of RGC death.
Moreover, reliable animal models are needed to assess the thera-
peutic value of drugs in preclinical trials (Smedowski et al., 2014).
However, since differences in retinal anatomy and physiology be-
tween animal species exist, and the procedures used to obtain such
experimental models may vary, it is necessary to ascertain the
advantages and limitations of a particular model, and carefully
choose the pertinent one in function of the damage caused to the
optic nerve and the reproducibility (Smedowski et al., 2014; Vecino,
2008).
Neuroinflammation is a common aspect of many neurodegen-
erative diseases, including glaucoma, and microglia participate in
their pathology, although the precise extent to which they influ-
ence each disease is not yet clear. Microglial activation has been
observed both in eyes affected by ocular hypertension and in the
healthy contralateral eyes (Rojas et al., 2014), indicating complex
mechanisms are at play that involve both structural and diffusible
elements. Microglial fractalkine may be a key element of the
neurotoxicity exerted by microglia in an experimental model of
induced glaucoma since suppression of its CX3CR1 receptor
induced more extensive RGC death when IOP was elevated (Wang
et al., 2014). A relationship between the release of cytokines (IL-6,
IL-1, TNF-a, etc) and the initiation of apoptosis has been established
(Gonzalez-Scarano and Baltuch, 1999). The intervention of neuro-
toxic agents, alone or in collaboration, has been studied in vivo and
in vitro, in different animal models or in humans. Thus, while there
is abundant information, the immune system is complex, making it
difficult to define the main causes and the accompanying phe-
nomena. RGC death in glaucoma probably depends on the
concurrence of several stressors (for instance ROS, glutamate,
mitochondrial impairment…), as well as ill-tuned or adapted
cellular responses (Rieck, 2013). Thus, full resolution of this “puz-
zle” will require more effort.
Gene expression dependent on TGF-b2 activation in astrocytes
cultured from the optic nerve head is reduced after antioxidant
treatment, indicating that antioxidants may have a place in the
treatment of neurodegenerative diseases like glaucoma (Yu and
Welge-Lussen, 2013). Astrocytes undergo morphological changes
related to the damage produced in RGCs (Lye-Barthel et al., 2013).
Hypertrophia of astrocytes was observed in the optic nerve in a rat
in vivo model of transient intraocular hypertension, as was early
up-regulation of phosphorylated STAT3 (signal transducer and
activator of transcription protein 3), a key transcription factor that
regulates the expression of many genes and that may play a crucial
role in the activation of astrocytes after an increase in IOP (Zhang
et al., 2013). Indeed, it is proposed that the damage observed in
optic nerve axons after an increase in IOP is secondary to the
damage induced in astrocytes, which suggests they are the primary
site of damage in early stages of glaucoma (Dai et al., 2012a).
In a mouse model of hereditary glaucoma (DBA/2J), an increase
in BDNF in hypertrophic astrocytes was concomitant with a deficit
in anterograde axonal transport prior to RGC synapse loss (Crish
et al., 2013). Hence, astrocytes may respond to the diminished
axonal transport by taking up BDNF from target neurons to mini-
mize the damage. It is very possible that the same mechanism will
work at the onset of glaucoma and that after the first days of high
IOP, the homeostatic mechanisms in the retina will be altered and
glial cells will then orchestrate the events that will provoke the
death of certain RGCs. It is unclear why not all RGCs die at the same
time in Glaucoma, however, the glial environment of the vulnerable
RGCs may be different and thus, these RGCs might respond to the
malevolent signals in a more aggressive manner.
In rats where glaucoma was induced by cauterization of epis-
cleral veins, in addition to RGC death changes in amacrine and bi-
polar cells were found, as well as astrocyte activation in the
damaged but not the contralateral eye (Hernandez et al., 2010). The
function of astrocyte and Müller cell activation is unknown as yet,
although one week after the IOP elevation we observe more
astrocyte gliosis that decreased thereafter (Hernandez et al., 2010).
It is possible that the astrocytes detect the increase in pressure
directly, or through microglial or Müller cells, and they react by
liberating neurotrophic signals that could help RGCs to resist the
insult. It is also possible that glial cells may provide mechanical
support. Another possible function of reactive astrocytes might be
related to repairing the damage to the blood retina barrier (Bush
et al., 1999; Voskuhl et al., 2009). However, the detrimental side
of astrogliosis is that reactive astrocytes may potentially exacerbate
the glaucomatous process by overexpressing toxins, producing a
direct toxic effect on RGCs. Müller cells overexpress GS, especially
in their inner and outer regions, while the vimentin cytoskeleton is
also partially disorganized (Fig. 14). AQP9 expression is reduced in
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40 29

Fig. 14. Glial cells in glaucomatous retinas. Comparative distribution of glial cells between control (A, C, E) and glaucomatous (B, D, F) rat retinas. A and B are whole-mount retinas
where the astrocytes were labelled with antibodies against-GFAP (green). Note the change in astrocyte morphology in the glaucoma retina (in B) compared to the control (A).
Vimentin (C, D) and glutamine synthetase (E, F) immunoreactivity (red) is evident in Müller cells. Nuclei were DAPI labelled (blue). Note the change in vimentin expression that is
more disorganized (D) than in the control eye (C) and GS expression is increased in F with respect to the control eye in E. Scale bar ¼ 50 mm.

RGCs of glaucomatous eyes, while glaucoma induces AQP7
expression in the Müller cell endfeet. Alternatively, the expression
of both AQP7 and AQP9 is reduced in the ciliary body of glaucom-
atous eyes. Therefore, AQP expression seems to play some role in
glaucoma (Tran et al., 2014).
Under normal conditions, resident macrophages maintain tis-
sue homeostasis by removing dead cells and other debris, and by
producing growth factors. However, under noxious conditions the
cellular stress response is activated that results in cell-
autonomous adaption. However, communication between
stressed cells and other cells in a tissue may also occur, including
resident macrophages. If the nature or the extent of stress is such
that it affects not only individual cells but also the entire tissue
(e.g. hypoxia or hyperthermia), then a tissue-level stress response
or parainflammation is elicited by the resident macrophages.
Depending on the strength of the noxious conditions, this
response can involve the recruitment of different types of circu-
lating inflammatory monocytes. Parainflammation may also
involve the mild release of plasma components into the affected
tissue and if the condition is sufficiently severe (e.g., infection or
injury), an acute inflammatory response ensues. This is charac-
terized by the recruitment of neutrophils and specialized subsets
of monocytes from the circulation that help to protect the host
from infection, promoting tissue repair and restoration of ho-
meostasis (Medzhitov, 2008, 2010). This could be the case of
experimental damage to the eye where strong inflammation could
activate resident cells and the immune system. Since astrocytes
and microglial cells have receptors for inflammation, this could
explain the contralateral response to damage in experimental
models of hypertension when laser damage causes intense
inflammation of the eye. It could also be relevant to situations in
humans where inflammation of different sources (uveitis, trauma,
etc…) could activate the immune system and affect the contra-
lateral eye. We must pay close attention to the different experi-
mental models to induce hypertension and glaucoma (Ruiz-
Ederra et al., 2005; Vecino and Sharma, 2011).
30 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
6.3. Age-related macular degeneration (AMD)
Advanced age is a risk factor for numerous pathologies, collab-
orating with other intrinsic and extrinsic cues that provoke the
initiation of a given disease. The brain and the retina suffer age-
related deterioration/degeneration (Harry, 2013), and to discover
the importance of age in this process, which cannot be avoided or
changed, it is paramount to unravel the influence of the other main
aetiological elements (for example life-style and environmental
factors) on the initiation and progression of neurodegeneration.
Microglia populate the retina early in the embryonic period and
their persistence during adulthood depends on self-renewal
through multiple divisions (Ginhoux et al., 2010). Microglial sen-
esce has been observed in healthy aging rats in conjunction with
telomere shortening and reduced telomerase activity (Flanary et al.,
2007). Other observations indicate that old microglia suffer age-
related alterations in their dynamic capacities (motility, branch-
ing, length of processes, etc.) and that they are less responsive to
external stimuli like ATP (Damani et al., 2011). Hence, the homeo-
static role of microglia can be seriously altered with age (Karlstetter
and Langmann, 2014).
AMD is a neurodegenerative disorder in which age plays a
principal role. The most common form of this disease is the atro-
phic or “dry” form that is characterized by RPE degeneration and
central retina photoreceptor death. In the exudative or “wet” form,
choroidal neo-vascularization is observed (Ma et al., 2009; Zhao
et al., 2014b). In healthy retinas, microglia are located in the inner
retina and they are scarce or non-existent in the outer retina and
retino-choroidal zone. However, in AMD the distribution of
microglia changes dramatically. Activated microglia migrate to sub-
retinal locations and in some cases, they come into close contact
with drusen deposits (Ma et al., 2009, 2012). In an in vitro model
where microglial cells were co-cultivated with primary RPE cells
and in an in vivo mouse model where microglia were transplanted
into the sub-retinal space, microglia appear to be responsible for
early alterations to RPE cells, and the initiation of a pro-
inflammatory and pro-angiogenic environment (Ma et al., 2009).
Therefore, the translocation of microglia to the outer retina may be
an essential event that triggers chronic inflammation related to the
progress of AMD.
Genetic models provide valuable information to analyse the
pathology of age-related neurodegeneration and to study the
participation of microglia in such processes (Karlstetter et al., 2010).
For example, knock-out mice develop some characteristics of AMD
at old ages, such as Ccl2 (CCL2 is a chemokine) and Cx3cr1 deficient
animals, and the Ccl2/Cx3cr1 double knock-out developed evident
lesions consistent with AMD earlier: RPE atrophy, photoreceptor
death, neovascularisation, and migration of microglia to the outer
nuclear layer and sub-retinal spaces (Tuo et al., 2007). In a knock-
out mice deficient in Nrf2 (nuclear erythroid 2-related factor e a
transcription factor that regulates the expression of protective
proteins against oxidative stress), the abnormal presence of
microglia, regulatory T cells and IL-17 (interleukin 17) producing T
cells in the retina and at sub-retinal locations has been documented
(Zhao et al., 2014b). Therefore, the destruction of the RPE in early
AMD favours the infiltration of circulating blood cells that
contribute significantly to the pathology and progression of AMD.
The AhR (a nuclear aryl hydrocarbon receptor) signalling pathway
is a defence mechanism that detoxifies metabolic waste molecules
and xenobiotics, and its activity in human RPE cells diminishes with
age (Hu et al., 2013). A knock-out mouse model deficient in AhR
(Ahr/) exhibits phenotypic characteristics of AMD, indicating that
this signalling pathway may hinder the appearance of AMD (Hu
et al., 2013; Kim et al., 2014). Accordingly, a complex collabora-
tion of mechanisms dependent on immune competent cells and
RPE cells appears to be responsible for the initiation and progres-
sion of AMD.
6.4. Retinitis pigmentosa (RP)
RP is a group of heterogeneous inherited diseases in which the
primary defect affects the retina, alone or in association to other
anomalies affecting different organs. RP is characterized by
photoreceptor degeneration and progressive blindness, yet the
molecular and cellular mechanisms involved in photoreceptor
death are not completely understood. Only when the mechanisms
underlying this damage are known can effective treatments follow.
As in other retinal pathologies, inflammation plays a fundamental
role in RP and it has been associated with mutations that affect
more than 70 genes related to different retinal functions (photo-
transduction, cell cycle proteins, ciliary proteins, structural pro-
teins, transcription factors, see RetNet at https://sph.uth.edu/
retnet/disease.htm). However, in some cases the genetic defect is
yet to be defined. Recently, a dominant mutation in the gene that
encodes HK1 (hexokinase 1, an enzyme that catalyzes the phos-
phorylation of glucose in the glucolytic pathway) was found to
cause RP (Sullivan et al., 2014).
Microglia sense the environment and they rapidly respond to
disturbances by trying to protect neurons against insults (neuro-
protection). However, the persistence of damage may activate the
neurotoxic phenotype of microglia, in an attempt to limit the
damage (Langmann, 2007). The early intervention of microglia in
degeneration suggests that they play an active role in pathological
mechanisms. In vitro and in vivo studies have characterized
microglial behaviour in RP and other degenerative retinal diseases
by studying their temporal transcription profiles, secretomes,
phenotypic changes and migration (Karlstetter et al., 2010;
Langmann, 2007). Using genetic mouse models and in vitro ap-
proaches, the complex and multiple effects of microglia as key el-
ements in RP and other examples of inherited retinal degeneration
can be illustrated. An analysis of microglia transcripts and
morphology in a retinoschisin-deficient mouse that suffers a severe
photoreceptor dystrophy indicated that microglia act in early stages
of the diseases (Gehrig et al., 2007; Karlstetter et al., 2010). In a
mouse RP model, rd (carrying a spontaneous defect in the b subunit
of rod cGMP-phosphodiesterase), early activation of microglia was
again seen by analysing the expression of microglial chemokine
transcripts and the secretion of TNF-a, which appeared prior to or
concomitant with photoreceptor death (Zeng et al., 2005).
Microglia-mediated neurotoxicity in photoreceptors is also sup-
ported by the activation of microglial NADPH oxidase, which gen-
erates ROS (Zeng et al., 2014). When the rd10 mouse model of RP is
crossed with a Cx3cr1GFP/GFP mouse, microglial activation was
evident as an early phenomenon in RP and Cx3cl1/Cx3cr1 signalling
significantly ameliorated microglia neurotoxicity (Peng et al., 2014).
Moreover, administration of minocycline for several days inhibited
microglia activation, reducing photoreceptor death and improved
vision in rd10 mice (Peng et al., 2014). Chemical destruction of
microglia in rd10 mouse retinal explants with clodronate-
containing liposomes slowed photoreceptor death, yet the neuro-
protective effect of IGF-I was attenuated when microglia were
depleted. These findings suggest a misbalance in the microglial
neuroprotective/neurotoxic intervention in this retinal dystrophy
(Arroba et al., 2011, 2014). In a rat model of RP bearing a mutation in
the rhodopsin-encoding gene (P23H), microglial cells were more
abundant in all retinal layers and they were also present in the sub-
retinal space. Treatment of these rats with taurodeoxycholic acid
prevented microglial activation and delayed photoreceptor death
(Noailles et al., 2014).
Not only are microglial cells activated in RP but Müller cells are
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40 31

more activated in the early stages of the disease (young animals) in receptor was expressed in Müller cells and it increased in the
a natural model of RP in dogs, the RP65 mutant. Müller cells also mutant retina, with expression in radial Müller fibres and sur-
respond to the ongoing disease process, although this response was rounding RGCs. Both vimentin and GFAP expression also increased,
not uniform against all the proteins examined. The low-affinity p75 although more prominently in younger animals than in adults. By

Fig. 15. Retinitis pigmentosa (RP). Comparison of the glia in controls (A, D, G, J) and adult dog models of RP, RPE65/ (B, E, H, K) and adult dog RPE65/ treated with adeno-
associated virus (AAV) vector containing a functional copy of the RPE65 gene (AAV-RPE65: C, F, I, L). (AeC) RGCs are labelled with antibodies against-BDNF (green) and the
Müller cells are stained with antibodies against p75 (red). The BDNF expression in RGCs decreased in affected retinas and increased after treatment. (DeF) GFAP expression (red)
increased in affected dogs and RPE65 expression (green) is lost in affected animals but it is recovered in treated dogs. (GeI) CRALBP was expressed in Müller cells (green) and it
appears disorganized in the affected and in the treated dogs. Increased expression of CRALBP was found in RPE in affected dogs (H) and it returned to control levels in gene-therapy
treated dogs (I). (JeL) Vimentin is expressed in Müller cells (red, DeF), and it was enhanced in the affected (K) and treated (L) dogs. Nuclei were stained with DAPI (blue). Scale
bar ¼ 50 mm.
32 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
contrast, CRALBP did not show an initial increase and it was
expressed below control levels in adult mutant dogs. It is clear that
Müller cells respond to outer retinal disease. The response was
mild, transient and more intense in the younger than in the older
mutant retinas, possibly indicating a lack of progressive retinal
degenerative changes in the period analyzed. However, only a
limited number of antibodies were analysed, two of which were
directed at IFs. It is possible that other proteins (e.g., GC and car-
bonic anhydrase C) would respond differently as they are known to
decrease in experimental retinal detachment in adult cats (Lewis
et al., 1994).
Therapeutic approaches in RP and other hereditary diseases
involving retinal degeneration might aim to restore the gene defect
by gene-replacement (Beltran et al., 2014) or stem-cell trans-
plantation (He et al., 2014). Retinal degeneration might also be
palliated or slowed by controlling the neuroinflammatory frame-
work. This can be achieved with either immune-suppressant or
immunomodulatory drugs (Karlstetter et al., 2010). Another strat-
egy includes the administration of protective molecules, such as
rod-derived cone viability factors, to preserve cone-dependent
central vision after rod death (Leveillard et al., 2014). In addition,
the application of sub-retinal photovoltaic arrays that restore
photoreceptor function can be applied when the inner retinal cir-
cuits are preserved (Light et al., 2014). Indeed different therapeutic
approaches should target the specific features of each type of RP
and hereditary retinal degeneration.
In diseases where the outer part of the retina is affected,
Müller cells sense the damage in the outer retina and transmit the
information to the inner retina. This event can be studied in dogs
carrying a mutation similar to Leber Congenital Amaurosis (LCA),
a specific type of RP. This disease comprises a group of childhood-
onset, autosomal recessive retinal diseases that result in severe
visual impairment or blindness (den Hollander et al., 2008). LCA
is caused by mutations in the RPE65 gene, which encodes the
65 kDa RPE-specific isomerase involved in visual pigment
regeneration (Gu et al., 1997). The RPE65 protein is essential to
maintain retinal function and photoreceptor viability, and for
retinoid cycling between the RPE and photoreceptors (Saari,
2000). Mutations in RPE65 occur naturally in dogs (Aguirre
et al., 1998), and gene therapy studies in these dogs (Acland
et al., 2001) has led to the first gene therapy undertaken in
humans (Hauswirth et al., 2008). We studied the molecular
changes that take place in the retina of the mutant dogs as they
mature (Hernandez et al., 2010) and found activation of astrocytes
in the GC layer, even when the damage to the retina originated in
the RPE. Hence, Müller cells appear to sense the changes in the
outer part of the retina and communicate these to cells located in
the inner part of the retina. Moreover, astrocytes located in the
neighbourhood of RGCs may also sense that synaptic trans-
mission was disturbed and react to this malfunction. Interest-
ingly, after gene therapy with an adeno-associated virus to
restore the mutated RP65 gene (AAV_RPE65), and even when
functional recovery was achieved (Acland et al., 2001), the glial
cells remained activated 6e24 months after treatment (Fig. 15).
Only the CRALBP immunoreactivity in the RPE returned to normal
levels after gene therapy, while Müller cells remained activated,
as evident by examining vimentin labelling. Müller cells can
change the morphology of their membranes, adapting to the
changes in the damaged cells surrounding them. After this
damage and retinal function is restored, as is this case with this
gene therapy, Müller cells should return to the original state in a
plastic-like process, although has yet to be confirmed (after the
submission of this review and during the process of revision a
book on retinal glia appeared where details of each cell type are
addressed: Reichenbach and Bringmann, 2015).
7. Conclusions, remarks and future directions
The retina contains three major classes of glia, Müller cells, as-
trocytes and a resident subpopulation of microglial cells. All are
integrated with the neurons that form the retina and allow a correct
functioning of the retina. Müller cells, astrocytes and microglia are
all capable of phagocytosis, responding to immunological damage
and they interact with neurons, thereby modulating the synapses.
Moreover, the three cell types play a role in the metabolism of
neurons, which is affected by their distribution and physiological
state. Since the neurons in the retina are highly specialized, their
metabolic demands are also very specific, and glial cells sur-
rounding these neurons provide different nutrients. Therefore, it is
crucial that when exploring the changes within the retina, an in-
tegrated approach should be adopted that considers glialeneuron
interactions, in order to fully understand the retina in health and
disease.
The source and age of glia is an important factor when deter-
mining the support that astrocytes or Müller glia can offer to RGCs.
Much of the research concerning the molecular mechanisms of RGC
processing in association with retinal glia has been carried out
using embryonic tissue, the rational being that axon outgrowth
during development could provide insight into the deficits under-
lying unsuccessful adult regeneration. Whilst much information
can be gleaned from these studies, there are limitations to the
comparisons that can be made, since there is a wealth of evidence
showing that adult RGCs and glia have a highly altered molecular
repertoire when compared to their embryonic and postnatal
counterparts. Therefore, it is imperative that attention is paid to
study adult RGCs and adult retinal glia.
The other limitation to studies in early periods is the use of
astrocytes from non-retinal sources. We are now only just begin-
ning to understand the complexity of glial heterogeneity and the
roles of different subpopulations within a single tissue, and further
research on the adult retina is required to explore the possibility
that some populations of retinal glia are more supportive of RGCs
than others. The molecular changes that take place in the adult
retina after damage occur rapidly, within minutes or hours. How-
ever, the return to a normal situation can take years, although some
restoration of function can be achieved in a shorter period.
Acknowledgements
We acknowledge the support of The Glaucoma Foundation
(TGF), Fundacio
n ONCE (name of the price: International price
ONCE IþD in biomedicine and new technologies for blind people
2004), Fundaluce and the Grupos Consolidados del Gobierno Vasco
(IT437-10) to E.V., and that of the IKERBASKE, Basque Foundation of
Science, Bilbao, Spain to S.C.S.
References
Acland, G.M., Aguirre, G.D., Ray, J., Zhang, Q., Aleman, T.S., Cideciyan, A.V., Pearce-
Kelling, S.E., Anand, V., Zeng, Y., Maguire, A.M., Jacobson, S.G., Hauswirth, W.W.,
Bennett, J., 2001. Gene therapy restores vision in a canine model of childhood
blindness. Nat. Genet. 28, 92e95.
Adijanto, J., Du, J., Moffat, C., Seifert, E.L., Hurle, J.B., Philp, N.J., 2014. The retinal
pigment epithelium utilizes fatty acids for ketogenesis. J. Biol. Chem. 289,
20570e20582.
Agte, S., Junek, S., Matthias, S., Ulbricht, E., Erdmann, I., Wurm, A., Schild, D., Kas, J.A.,
Reichenbach, A., 2011. Muller glial cell-provided cellular light guidance through
the vital guinea-pig retina. Biophys. J. 101, 2611e2619.
Aguirre, G.D., Baldwin, V., Pearce-Kelling, S., Narfstrom, K., Ray, K., Acland, G.M.,
1998. Congenital stationary night blindness in the dog: common mutation in
the RPE65 gene indicates founder effect. Mol. Vis. 4, 23.
Ahonen, S.J., Kaukonen, M., Nussdorfer, F.D., Harman, C.D., Komaromy, A.M., Lohi, H.,
2014. A novel Missense mutation in ADAMTS10 in Norwegian Elkhound pri-
mary glaucoma. PLoS One 9, e111941.
Ai, J., Liu, Y., Sun, J.H., 2013. Advanced glycation end-products stimulate basic
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
fibroblast growth factor expression in cultured Muller cells. Mol. Med. Rep. 7,
16e20.
Ajami, B., Bennett, J.L., Krieger, C., Tetzlaff, W., Rossi, F.M., 2007. Local self-renewal
can sustain CNS microglia maintenance and function throughout adult life.
Nat. Neurosci. 10, 1538e1543.
Akhter, N., Nix, M., Abdul, Y., Singh, S., Husain, S., 2013. Delta-opioid receptors
attenuate TNF-alpha-induced MMP-2 secretion from human ONH astrocytes.
Invest. Ophthalmol. Vis. Sci. 54, 6605e6611.
Allen, N.J., Bennett, M.L., Foo, L.C., Wang, G.X., Chakraborty, C., Smith, S.J.,
Barres, B.A., 2012. Astrocyte glypicans 4 and 6 promote formation of excitatory
synapses via GluA1 AMPA receptors. Nature 486, 410e414.
Amadio, S., De Ninno, A., Montilli, C., Businaro, L., Gerardino, A., Volonte, C., 2013.
Plasticity of primary microglia on micropatterned geometries and spontaneous
long-distance migration in microfluidic channels. BMC Neurosci. 14, 121e2202,
2214-2121.
Amaratunga, A., Abraham, C.R., Edwards, R.B., Sandell, J.H., Schreiber, B.M., Fine, R.E.,
1996. Apolipoprotein E is synthesized in the retina by Muller glial cells, secreted
into the vitreous, and rapidly transported into the optic nerve by retinal gan-
glion cells. J. Biol. Chem. 271, 5628e5632.
Anderson, D.H., Hageman, G.S., Mullins, R.F., Neitz, M., Neitz, J., Ozaki, S.,
Preissner, K.T., Johnson, L.V., 1999. Vitronectin gene expression in the adult
human retina. Invest. Ophthalmol. Vis. Sci. 40, 3305e3315.
Anderson, D.R., Hoyt, W.F., Hogan, M.J., 1967. The fine structure of the astroglia in
the human optic nerve and optic nerve head. Trans. Am. Ophthalmol. Soc. 65,
275e305.
Argaw, A.T., Gurfein, B.T., Zhang, Y., Zameer, A., John, G.R., 2009. VEGF-mediated
disruption of endothelial CLN-5 promotes blood-brain barrier breakdown. Proc.
Natl. Acad. Sci. U. S. A. 106, 1977e1982.
Arroba, A.I., Alvarez-Lindo, N., van Rooijen, N., de la Rosa, E.J., 2011. Microglia-
mediated IGF-I neuroprotection in the rd10 mouse model of retinitis pigmen-
tosa. Invest. Ophthalmol. Vis. Sci. 52, 9124e9130.
Arroba, A.I., Alvarez-Lindo, N., van Rooijen, N., de la Rosa, E.J., 2014. Microglia-
Muller glia crosstalk in the rd10 mouse model of retinitis pigmentosa. Adv. Exp.
Med. Biol. 801, 373e379.
Bachoo, R.M., Kim, R.S., Ligon, K.L., Maher, E.A., Brennan, C., Billings, N., Chan, S.,
Li, C., Rowitch, D.H., Wong, W.H., DePinho, R.A., 2004. Molecular diversity of
astrocytes with implications for neurological disorders. Proc. Natl. Acad. Sci. U.
S. A. 101, 8384e8389.
Bahr, M., Przyrembel, C., Bastmeyer, M., 1995. Astrocytes from adult rat optic nerves
are nonpermissive for regenerating retinal ganglion cell axons. Exp. Neurol. 131,
211e220.
Barres, B., Dowling, J., 2010. Roles of astrocytes and other glial cells in glaucoma and
other retinal diseases. Astrocytes Glaucomatous Neurodegener.
Barres, B.A., 1991. New roles for glia. J. Neurosci. 11, 3685e3694.
Barres, B.A., 2008. The mystery and magic of glia: a perspective on their roles in
health and disease. Neuron 60, 430e440.
Bartsch, U., Bartsch, S., Dorries, U., Schachner, M., 1992. Immunohistological local-
ization of tenascin in the developing and Lesioned adult mouse optic nerve. Eur.
J. Neurosci. 4, 338e352.
Bartsch, U., Faissner, A., Trotter, J., Dorries, U., Bartsch, S., Mohajeri, H.,
Schachner, M., 1994. Tenascin demarcates the boundary between the myelin-
ated and nonmyelinated part of retinal ganglion cell axons in the developing
and adult mouse. J. Neurosci. 14, 4756e4768.
Bates, C.A., Becker, C.G., Miotke, J.A., Meyer, R.L., 1999. Expression of polysialylated
NCAM but not L1 or N-cadherin by regenerating adult mouse optic fibers
in vitro. Exp. Neurol. 155, 128e139.
Bazan, N.G., 2007. Omega-3 fatty acids, pro-inflammatory signaling and neuro-
protection. Curr. Opin. Clin. Nutr. Metab. Care 10, 136e141.
Becker, S., Singhal, S., Jones, M.F., Eastlake, K., Cottrill, P.B., Jayaram, H., Limb, A.,
2013. Acquisition of RGC phenotype in human Müller glia with stem cell
characteristics is accompanied by upregulation of functional nicotinic acetyl-
choline receptors. Mol. Vis. 19, 1925e1936.
Belecky-Adams, T.L., Chernoff, E.C., Wilson, J.M., Dharmarajan, S., 2013. Reactive
muller glia as potential retinal progenitors. In: Bonfanti, L. (Ed.), Neural Stem
Cells e New Perspectives. InTech.
Beltran, W.A., Cideciyan, A.V., Lewin, A.S., Hauswirth, W.W., Jacobson, S.G.,
Aguirre, G.D., 2014. Gene Augmentation for X-linked retinitis pigmentosa
caused by mutations in RPGR. Cold Spring Harb. Perspect. Med.
Benveniste, E.N., 1993. Astrocyte-microgía interactions. In: Murphy, S. (Ed.), Astro-
cytes. Pharmacology and Function. Academic Press, San Diego, pp. 355e382.
Berger, U.V., Luthi-Carter, R., Passani, L.A., Elkabes, S., Black, I., Konradi, C., Coyle, J.T.,
1999. Glutamate carboxypeptidase II is expressed by astrocytes in the adult rat
nervous system. J. Comp. Neurol. 415, 52e64.
Besser, M., Jagatheaswaran, M., Reinhard, J., Schaffelke, P., Faissner, A., 2012.
Tenascin C regulates proliferation and differentiation processes during embry-
onic retinogenesis and modulates the de-differentiation capacity of Muller glia
by influencing growth factor responsiveness and the extracellular matrix
compartment. Dev. Biol. 369, 163e176.
Bhatti, F., Ball, G., Hobbs, R., Linens, A., Munzar, S., Akram, R., Barber, A.J.,
Anderson, M.P., Elliott, M.H., Edwards, M., 2014. Pulmonary surfactant protein A
is expressed in the mouse retina by muller cells and impacts neovascularization
in oxygen induced retinopathy. Invest. Ophthalmol. Vis. Sci.
Bignami, A., Eng, L.F., Dahl, D., Uyeda, C.T., 1972. Localization of the glial fibrillary
acidic protein in astrocytes by immunofluorescence. Brain Res. 43, 429e435.
Bilimoria, P.M., Stevens, B., 2014. Microglia function during brain development: new
33
insights from animal models. Brain Res.
Blackshaw, S., Harpavat, S., Trimarchi, J., Cai, L., Huang, H., Kuo, W.P., Weber, G.,
Lee, K., Fraioli, R.E., Cho, S.H., Yung, R., Asch, E., Ohno-Machado, L., Wong, W.H.,
Cepko, C.L., 2004. Genomic analysis of mouse retinal development. PLoS Biol. 2,
E247.
Bonnet, D., Garcia, M., Vecino, E., Lorentz, J.G., Sahel, J., Hicks, D., 2004. Brain-
derived neurotrophic factor signalling in adult pig retinal ganglion cell neurite
regeneration in vitro. Brain Res. 1007, 142e151.
Bosco, A., Inman, D.M., Steele, M.R., Wu, G., Soto, I., Marsh-Armstrong, N.,
Hubbard, W.C., Calkins, D.J., Horner, P.J., Vetter, M.L., 2008. Reduced retina
microglial activation and improved optic nerve integrity with minocycline
treatment in the DBA/2J mouse model of glaucoma. Invest. Ophthalmol. Vis. Sci.
49, 1437e1446.
Bosco, A., Linden, R., Carri, N.G., 1994. Expression of alpha-1 integrin subunit in the
mammalian retina. Cell Biol. Int. 18, 211e213.
Bossy, B., Bossy-Wetzel, E., Reichardt, L.F., 1991. Characterization of the integrin
alpha 8 subunit: a new integrin beta 1-associated subunit, which is promi-
nently expressed on axons and on cells in contact with basal laminae in chick
embryos. EMBO J. 10, 2375e2385.
Bradshaw, A.D., McNagny, K.M., Gervin, D.B., Cann, G.M., Graf, T., Clegg, D.O., 1995.
Integrin alpha 2 beta 1 mediates interactions between developing embryonic
retinal cells and collagen. Development 121, 3593e3602.
Bringmann, A., Grosche, A., Pannicke, T., Reichenbach, A., 2013. GABA and glutamate
uptake and metabolism in retinal glial (Muller) cells. Front. Endocrinol. (Lau-
sanne) 4, 48.
Bringmann, A., Iandiev, I., Pannicke, T., Wurm, A., Hollborn, M., Wiedemann, P.,
Osborne, N.N., Reichenbach, A., 2009a. Cellular signaling and factors involved in
Müller cell gliosis: neuroprotective and detrimental effects. Prog. Retin. Eye Res.
28, 423e451.
Bringmann, A., Pannicke, T., Biedermann, B., Francke, M., Iandiev, I., Grosche, J.,
Wiedemann, P., Albrecht, J., Reichenbach, A., 2009b. Role of retinal glial cells in
neurotransmitter uptake and metabolism. Neurochem. Int. 54, 143e160.
Bringmann, A., Pannicke, T., Grosche, J., Francke, M., Wiedemann, P., Skatchkov, S.N.,
Osborne, N.N., Reichenbach, A., 2006. Müller cells in the healthy and diseased
retina. Prog. Retin. Eye Res. 25, 397e424.
Bringmann, A., Wiedemann, P., 2012. Muller glial cells in retinal disease. Oph-
thalmologica 227, 1e19.
Bruckner, E., Grosche, A., Pannicke, T., Wiedemann, P., Reichenbach, A.,
Bringmann, A., 2012. Mechanisms of VEGF- and glutamate-induced inhibition
of osmotic swelling of murine retinal glial (Muller) cells: indications for the
involvement of vesicular glutamate release and connexin-mediated ATP
release. Neurochem. Res. 37, 268e278.
Buffo, A., Rite, I., Tripathi, P., Lepier, A., Colak, D., Horn, A.P., Mori, T., Gotz, M., 2008.
Origin and progeny of reactive gliosis: a source of multipotent cells in the
injured brain. Proc. Natl. Acad. Sci. U. S. A. 105, 3581e3586.
Bunt-Milam, A.H., Saari, J.C., 1983. Immunocytochemical localization of two
retinoid-binding proteins in vertebrate retina. J. Cell Biol. 97, 703e712.
Burda, J.E., Sofroniew, M.V., 2014. Reactive gliosis and the multicellular response to
CNS damage and disease. Neuron 81, 229e248.
Bush, T.G., Puvanachandra, N., Horner, C.H., Polito, A., Ostenfeld, T., Svendsen, C.N.,
Mucke, L., Johnson, M.H., Sofroniew, M.V., 1999. Leukocyte infiltration, neuronal
degeneration, and neurite outgrowth after ablation of scar-forming, reactive
astrocytes in adult transgenic mice. Neuron 23, 297e308.
Cahoy, J.D., Emery, B., Kaushal, A., Foo, L.C., Zamanian, J.L., Christopherson, K.S.,
Xing, Y., Lubischer, J.L., Krieg, P.A., Krupenko, S.A., Thompson, W.J., Barres, B.A.,
2008. A transcriptome database for astrocytes, neurons, and oligodendrocytes:
a new resource for understanding brain development and function. J. Neurosci.
28, 264e278.
Cann, G.M., Bradshaw, A.D., Gervin, D.B., Hunter, A.W., Clegg, D.O., 1996. Widespread
expression of beta1 integrins in the developing chick retina: evidence for a role
in migration of retinal ganglion cells. Dev. Biol. 180, 82e96.
Carson, M.J., Doose, J.M., Melchior, B., Schmid, C.D., Ploix, C.C., 2006. CNS immune
privilege: hiding in plain sight. Immunol. Rev. 213, 48e65.
Carulli, D., Laabs, T., Geller, H.M., Fawcett, J.W., 2005. Chondroitin sulfate pro-
teoglycans in neural development and regeneration. Curr. Opin. Neurobiol. 15,
116e120.
Casson, R.J., Chidlow, G., Han, G., Wood, J.P., 2013. An explanation for the Warburg
effect in the adult mammalian retina. Clin. Exp. Ophthalmol. 41, 517.
Cepko, C., 1993. Lineage versus environment in the embryonic retina. Trends
Neurosci. 16, 96e97. Author reply 98.
Chaboub, L.S., Deneen, B., 2012. Developmental origins of astrocyte heterogeneity:
the final frontier of CNS development. Dev. Neurosci. 34, 379e388.
Chai, L., Morris, J.E., 1999. Heparan sulfate in the inner limiting membrane of em-
bryonic chicken retina binds basic fibroblast growth factor to promote axonal
outgrowth. Exp. Neurol. 160, 175e185.
Chang, K.C., Ponder, J., Labarbera, D.V., Petrash, J.M., 2014. Aldose reductase inhi-
bition prevents endotoxin-induced inflammatory responses in retinal microglia.
Invest. Ophthalmol. Vis. Sci. 55, 2853e2861.
Chen, K., Zhang, Q., Wang, J., Liu, F., Mi, M., Xu, H., Chen, F., Zeng, K., 2009a. Taurine
protects transformed rat retinal ganglion cells from hypoxia-induced apoptosis
by preventing mitochondrial dysfunction. Brain Res. 1279, 131e138.
Chen, M., Forrester, J.V., Xu, H., 2007. Synthesis of complement factor H by retinal
pigment epithelial cells is down-regulated by oxidized photoreceptor outer
segments. Exp. Eye Res. 84, 635e645.
Chen, M., Muckersie, E., Luo, C., Forrester, J.V., Xu, H., 2010. Inhibition of the
34 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
alternative pathway of complement activation reduces inflammation in
experimental autoimmune uveoretinitis. Eur. J. Immunol. 40, 2870e2881.
Chen, M., Zhao, J., Luo, C., Pandi, S.P., Penalva, R.G., Fitzgerald, D.C., Xu, H., 2012.
Para-inflammation-mediated retinal recruitment of bone marrow-derived
myeloid cells following whole-body irradiation is CCL2 dependent. Glia 60,
833e842.
Chen, W., Mo, W., Sun, K., Huang, X., Zhang, Y.L., Song, H.Y., 2009b. Microplasmin
degrades fibronectin and laminin at vitreoretinal interface and outer retina
during enzymatic vitrectomy. Curr. Eye Res. 34, 1057e1064.
Chen, X., Zhou, H., Gong, Y., Wei, S., Zhang, M., 2014. Early spatiotemporal charac-
terization of microglial activation in the retinas of rats with streptozotocin-
induced diabetes. Graefes Arch. Clin. Exp. Ophthalmol.
Cherry, J.D., Olschowka, J.A., O'Banion, M.K., 2014. Neuroinflammation and M2
microglia: the good, the bad, and the inflamed. J. Neuroinflammation 11,
98e2094, 2011-2098.
Cho, K.S., Yang, L., Lu, B., Feng Ma, H., Huang, X., Pekny, M., Chen, D.F., 2005. Re-
establishing the regenerative potential of central nervous system axons in
postnatal mice. J. Cell Sci. 118, 863e872.
Christopherson, K.S., Ullian, E.M., Stokes, C.C., Mullowney, C.E., Hell, J.W., Agah, A.,
Lawler, J., Mosher, D.F., Bornstein, P., Barres, B.A., 2005. Thrombospondins are
astrocyte-secreted proteins that promote CNS synaptogenesis. Cell 120,
421e433.
Chung, W.S., Clarke, L.E., Wang, G.X., Stafford, B.K., Sher, A., Chakraborty, C., Joung, J.,
Foo, L.C., Thompson, A., Chen, C., Smith, S.J., Barres, B.A., 2013. Astrocytes
mediate synapse elimination through MEGF10 and MERTK pathways. Nature
504, 394e400.
Clark, S.J., Keenan, T.D., Fielder, H.L., Collinson, L.J., Holley, R.J., Merry, C.L., van
Kuppevelt, T.H., Day, A.J., Bishop, P.N., 2011. Mapping the differential distribu-
tion of glycosaminoglycans in the adult human retina, choroid, and sclera.
Invest. Ophthalmol. Vis. Sci. 52, 6511e6521.
Cohen, J., Burne, J.F., McKinlay, C., Winter, J., 1987. The role of laminin and the
laminin/fibronectin receptor complex in the outgrowth of retinal ganglion cell
axons. Dev. Biol. 122, 407e418.
Colton, C.A., 2009. Heterogeneity of microglial activation in the innate immune
response in the brain. J. Neuroimmune Pharmacol. 4, 399e418.
Coorey, N.J., Shen, W., Chung, S.H., Zhu, L., Gillies, M.C., 2012. The role of glia in
retinal vascular disease. Clin. Exp. Optom. 95, 266e281.
Copland, D.A., Hussain, K., Baalasubramanian, S., Hughes, T.R., Morgan, B.P., Xu, H.,
Dick, A.D., Nicholson, L.B., 2010. Systemic and local anti-C5 therapy reduces the
disease severity in experimental autoimmune uveoretinitis. Clin. Exp. Immunol.
159, 303e314.
Corraliza, I., 2014. Recruiting specialized macrophages across the borders to restore
brain functions. Front. Cell. Neurosci. 8, 262.
Crish, S.D., Dapper, J.D., MacNamee, S.E., Balaram, P., Sidorova, T.N., Lambert, W.S.,
Calkins, D.J., 2013. Failure of axonal transport induces a spatially coincident
increase in astrocyte BDNF prior to synapse loss in a central target. Neurosci-
ence 229, 55e70.
Cuenca, N., Fernandez-Sanchez, L., Campello, L., Maneu, V., De la Villa, P., Lax, P.,
Pinilla, I., 2014. Cellular responses following retinal injuries and therapeutic
approaches for neurodegenerative diseases. Prog. Retin. Eye Res. 43C, 17e75.
Dai, C., Khaw, P.T., Yin, Z.Q., Li, D., Raisman, G., Li, Y., 2012a. Structural basis of
glaucoma: the fortified astrocytes of the optic nerve head are the target of
raised intraocular pressure. Glia 60, 13e28.
Dai, M., Xia, X.B., Xiong, S.Q., 2012b. BDNF regulates GLAST and glutamine syn-
thetase in mouse retinal Müller cells. J. Cell. Physiol. 227, 596e603.
Damani, M.R., Zhao, L., Fontainhas, A.M., Amaral, J., Fariss, R.N., Wong, W.T., 2011.
Age-related alterations in the dynamic behavior of microglia. Aging Cell. 10,
263e276.
Danbolt, N.C., 2001. Glutamate uptake. Prog. Neurobiol. 65, 1e105.
Das, A.V., Mallya, K.B., Zhao, X., Ahmad, F., Bhattacharya, S., Thoreson, W.B.,
Hegde, G.V., Ahmad, I., 2006. Neural stem cell properties of Muller glia in the
mammalian retina: regulation by Notch and Wnt signaling. Dev. Biol. 299,
283e302.
Davalos, D., Grutzendler, J., Yang, G., Kim, J.V., Zuo, Y., Jung, S., Littman, D.R.,
Dustin, M.L., Gan, W.B., 2005. ATP mediates rapid microglial response to local
brain injury in vivo. Nat. Neurosci. 8, 752e758.
Davis, C.H., Kim, K.Y., Bushong, E.A., Mills, E.A., Boassa, D., Shih, T., Kinebuchi, M.,
Phan, S., Zhou, Y., Bihlmeyer, N.A., Nguyen, J.V., Jin, Y., Ellisman, M.H., Marsh-
Armstrong, N., 2014. Transcellular degradation of axonal mitochondria. Proc.
Natl. Acad. Sci. U. S. A. 111, 9633e9638.
Davis, E.J., Foster, T.D., Thomas, W.E., 1994. Cellular forms and functions of brain
microglia. Brain Res. Bull. 34, 73e78.
Davis, J.T., Wen, Q., Janmey, P.A., Otteson, D.C., Foster, W.J., 2012. Muller cell
expression of genes implicated in proliferative vitreoretinopathy is influenced
by substrate elastic modulus. Invest. Ophthalmol. Vis. Sci. 53, 3014e3019.
de Curtis, I., 1993. The alpha 6 beta 1 integrin is a laminin receptor for developing
retinal neurons. Cytotechnology 11 (Suppl. 1), S41eS43.
Deeg, C.A., Eberhardt, C., Hofmaier, F., Amann, B., Hauck, S.M., 2011. Osteopontin and
fibronectin levels are decreased in vitreous of autoimmune uveitis and retinal
expression of both proteins indicates ECM re-modeling. PLoS One 6, e27674.
Deguchi, J., Yamamoto, A., Fujiki, Y., Uyama, M., Tsukahara, I., Tashiro, Y., 1992.
Localization of nonspecific lipid transfer protein (nsLTP ¼ sterol carrier protein
2) and acyl-CoA oxidase in peroxisomes of pigment epithelial cells of rat retina.
J. Histochem. Cytochem. 40, 403e410.
Del Rio, P., Irmler, M., Arango-Gonzalez, B., Favor, J., Bobe, C., Bartsch, U., Vecino, E.,
Beckers, J., Hauck, S.M., Ueffing, M., 2011. GDNF-induced osteopontin from
Muller glial cells promotes photoreceptor survival in the Pde6brd1 mouse
model of retinal degeneration. Glia 59, 821e832.
Del Rio-Hortega, P., 1939. The microglia. Lancet 233, 1023e1926.
den Hollander, A.I., Roepman, R., Koenekoop, R.K., Cremers, F.P., 2008. Leber
congenital amaurosis: genes, proteins and disease mechanisms. Prog. Retin. Eye
Res. 27, 391e419.
Derouiche, A., Rauen, T., 1995. Coincidence of L-glutamate/L-aspartate transporter
(GLAST) and glutamine synthetase (GS) immunoreactions in retinal glia: evi-
dence for coupling of GLAST and GS in transmitter clearance. J. Neurosci. Res.
42, 131e143.
Dharmarajan, S., Gurel, Z., Wang, S., Sorenson, C.M., Sheibani, N., Belecky-
Adams, T.L., 2013. Bone morphogenetic protein 7 regulates reactive gliosis in
retinal astrocytes and Müller glia. Mol. Vis. 20, 1085e1108.
Ding, D., Xu, L., Xu, H., Li, X., Liang, Q., Zhao, Y., Wang, Y., 2014. Mash1 efficiently
reprograms rat astrocytes into neurons. Neural Regen. Res. 9, 25e32.
Dreher, Z., Robinson, S.R., Distler, C., 1992. Muller cells in vascular and avascular
retinae: a survey of seven mammals. J. Comp. Neurol. 323, 59e80.
Duband, J.L., Belkin, A.M., Syfrig, J., Thiery, J.P., Koteliansky, V.E., 1992. Expression of
alpha 1 integrin, a laminin-collagen receptor, during myogenesis and neuro-
genesis in the avian embryo. Development 116, 585e600.
Dvoriantchikova, G., Ivanov, D., 2014. Tumor necrosis factor-alpha mediates acti-
vation of NF-kappaB and JNK signaling cascades in retinal ganglion cells and
astrocytes in opposite ways. Eur. J. Neurosci. 40, 3171e3178.
Edelstein, L., Smythies, J., 2014. The role of epigenetic-related codes in neuro-
computation: dynamic hardware in the brain. Philos. Trans. R. Soc. Lond. B Biol.
Sci. 369.
Eichler, W., Yafai, Y., Wiedemann, P., Reichenbach, A., 2004. Angiogenesis related
factors derived from retinal glial (Müller) cells in hypoxia. Neuroreport 15,
1633e1637.
Ellis-Behnke, R.G., Jonas, R.A., Jonas, J.B., 2013. The microglial system in the eye and
brain in response to stimuli in vivo. J. Glaucoma 22 (Suppl. 5), S32eS35.
Enger, R., Gundersen, G.A., Haj-Yasein, N.N., Eilert-Olsen, M., Thoren, A.E.,
Vindedal, G.F., Petersen, P.H., Skare, O., Nedergaard, M., Ottersen, O.P.,
Nagelhus, E.A., 2012. Molecular scaffolds underpinning macroglial polarization:
an analysis of retinal Muller cells and brain astrocytes in mouse. Glia 60,
2018e2026.
Ennis, S., Kunz, Y.W., 1986. Differentiated retinal Muller glia are cil-
iatedeultrastructural evidence in the teleost Poecilia reticulata P. Cell Biol. Int.
Rep. 10, 611e622.
Erickson, P.A., Fisher, S.K., Guerin, C.J., Anderson, D.H., Kaska, D.D., 1987. Glial
fibrillary acidic protein increases in Muller cells after retinal detachment. Exp.
Eye Res. 44, 37e48.
Escartin, C., Brouillet, E., Gubbellini, P., Trioulier, Y., Jacquard, C., 2006. Ciliary
neurotrophic factor activates astrocytes, redistributes their glutamate trans-
porters GLAST and GLT-1 to raft microdomains, and improves glutamate
handling in vivo. J. Neurosci. 26, 5978e5989.
Escartin, C., Pierre, K., Colin, A., Brouillet, E., Delzescaux, T., 2007. Activation of as-
trocytes by CNTF induces metabolic plasticity and increases resistance to
metabolic insults. J. Neurosci. 27, 7094e7104.
Fawcett, J.W., Housden, E., Smith-Thomas, L., Meyer, R.L., 1989. The growth of axons
in three-dimensional astrocyte cultures. Dev. Biol. 135, 449e458.
Fernandes, A., Miller-Fleming, L., Pais, T.F., 2014. Microglia and inflammation:
conspiracy, controversy or control? Cell. Mol. Life Sci. 71, 3969e3985.
Fern
andez, J.M., Di Giusto, G., Kalstein, M., Melamud, L., Rivarola, V., Ford, P.,
Capurro, C., 2013. Cell volume regulation in cultured human retinal Müller cells
is associated with changes in transmembrane potential. PLoS One 8, e57268.
Fischer, A.J., Bongini, R., 2010. Turning Muller glia into neural progenitors in the
retina. Mol. Neurobiol. 42, 199e209.
Fischer, A.J., Reh, T.A., 2001. Muller glia are a potential source of neural regeneration
in the postnatal chicken retina. Nat. Neurosci. 4, 247e252.
Fischer, A.J., Schmidt, M., Omar, G., Reh, T.A., 2004. BMP4 and CNTF are neuro-
protective and suppress damage-induced proliferation of Muller glia in the
retina. Mol. Cell. Neurosci. 27, 531e542.
Flanary, B.E., Sammons, N.W., Nguyen, C., Walker, D., Streit, W.J., 2007. Evidence that
aging and amyloid promote microglial cell senescence. Rejuvenation Res. 10,
61e74.
Fletcher, E.L., Phipps, J.A., Wilkinson-Berka, J.L., 2005. Dysfunction of retinal neu-
rons and glia during diabetes. Clin. Exp. Optom. 88, 132e145.
Fliesler, S.J., Keller, R.K., 1997. Isoprenoid metabolism in the vertebrate retina. Int. J.
Biochem. Cell. Biol. 29, 877e894.
Foo, L.C., Allen, N.J., Bushong, E.A., Ventura, P.B., Chung, W.S., Zhou, L., Cahoy, J.D.,
Daneman, R., Zong, H., Ellisman, M.H., Barres, B.A., 2011. Development of a
method for the purification and culture of rodent astrocytes. Neuron 71,
799e811.
Formichella, C.R., Abella, S.K., Sims, S.M., Cathcart, H.M., Sappington, R.M., 2014.
Astrocyte reactivity: a biomarker for retinal ganglion cell health in retinal
neurodegeneration. J. Clin. Cell. Immunol. 5.
Franze, K., Francke, M., Guenter, K., Christ, A.F., Koerber, N., Reichenbach, A., Guck, J.,
2011. Spatial mapping of the mechanical properties of the living retina using
scanning force microscopy. Soft Matter 7, 3147e3154.
Franze, K., Grosche, J., Skatchkov, S.N., Schinkinger, S., Foja, C., Schild, D.,
Uckermann, O., Travis, K., Reichenbach, A., Guck, J., 2007. Müller cells are living
optical fibers in the vertebrate retina. Proc. Natl. Acad. Sci. U. S. A. 104,
8287e8292.
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
Frasson, M., Sahel, J.A., Fabre, M., Simonutti, M., Dreyfus, H., Picaud, S., 1999. Reti-
nitis pigmentosa: rod photoreceptor rescue by a calcium-channel blocker in the
rd mouse. Nat. Med. 5, 1183e1187.
Fuchshofer, R., Welge-Lussen, U., Lutjen-Drecoll, E., Birke, M., 2006. Biochemical and
morphological analysis of basement membrane component expression in cor-
neoscleral and cribriform human trabecular meshwork cells. Invest. Oph-
thalmol. Vis. Sci. 47, 794e801.
Galdos, M., Vecino, E., 2011. Ultrastructural changes in the trabecular meshwork and
increased IOP. Which came first, the chicken or the egg? Arch. Soc. Esp. Oftal-
mol. 86, 241e242.
Gallego, B.I., Salazar, J.J., de Hoz, R., Rojas, B., Ramirez, A.I., Salinas-Navarro, M.,
Ortin-Martinez, A., Valiente-Soriano, F.J., Aviles-Trigueros, M., Villegas-
Perez, M.P., Vidal-Sanz, M., Trivino, A., Ramirez, J.M., 2012. IOP induces upre-
gulation of GFAP and MHC-II and microglia reactivity in mice retina contra-
lateral to experimental glaucoma. J. Neuroinflammation 9, 92.
Garcia, M., Forster, V., Hicks, D., Vecino, E., 2002. Effects of muller glia on cell sur-
vival and neuritogenesis in adult porcine retina in vitro. Invest. Ophthalmol. Vis.
Sci. 43, 3735e3743.
Garcia, M., Forster, V., Hicks, D., Vecino, E., 2003. In vivo expression of neuro-
trophins and neurotrophin receptors is conserved in adult porcine retina
in vitro. Invest. Ophthalmol. Vis. Sci. 44, 4532e4541.
Garcia, M., Vecino, E., 2003. Role of Muller glia in neuroprotection and regeneration
in the retina. Histol. Histopathol. 18, 1205e1218.
Garcia, T.B., Pannicke, T., Vogler, S., Berk, B.A., Grosche, A., Wiedemann, P., Seeger, J.,
Reichenbach, A., Herculano, A.M., Bringmann, A., 2014. Nerve growth factor
inhibits osmotic swelling of rat retinal glial (Muller) and bipolar cells by
inducing glial cytokine release. J. Neurochem. 131, 303e313.
Garcia-Valenzuela, E., Shareef, S., Walsh, J., Sharma, S.C., 1995. Programmed cell
death of retinal ganglion cells during experimental glaucoma. Exp. Eye Res. 61,
33e44.
Garcia-Valenzuela, E., Sharma, S.C., Pina, A.L., 2005. Multilayered retinal microglial
response to optic nerve transection in rats. Mol. Vis. 11, 225e231.
Gehrig, A., Langmann, T., Horling, F., Janssen, A., Bonin, M., Walter, M., Poths, S.,
Weber, B.H., 2007. Genome-wide expression profiling of the retinoschisin-
deficient retina in early postnatal mouse development. Invest. Ophthalmol.
Vis. Sci. 48, 891e900.
Gertig, U., Hanisch, U.K., 2014. Microglial diversity by responses and responders.
Front. Cell. Neurosci. 8, 101.
Ginhoux, F., Greter, M., Leboeuf, M., Nandi, S., See, P., Gokhan, S., Mehler, M.F.,
Conway, S.J., Ng, L.G., Stanley, E.R., Samokhvalov, I.M., Merad, M., 2010. Fate
mapping analysis reveals that adult microglia derive from primitive macro-
phages. Science 330, 841e845.
Ginhoux, F., Lim, S., Hoeffel, G., Low, D., Huber, T., 2013. Origin and differentiation of
microglia. Front. Cell. Neurosci. 7, 45.
Glanzer, J.G., Enose, Y., Wang, T., Kadiu, I., Gong, N., Rozek, W., Liu, J.,
Schlautman, J.D., Ciborowski, P.S., Thomas, M.P., Gendelman, H.E., 2007.
Genomic and proteomic microglial profiling: pathways for neuroprotective
inflammatory responses following nerve fragment clearance and activation.
J. Neurochem. 102, 627e645.
Goldmann, T., Wieghofer, P., Muller, P.F., Wolf, Y., Varol, D., Yona, S., Brendecke, S.M.,
Kierdorf, K., Staszewski, O., Datta, M., Luedde, T., Heikenwalder, M., Jung, S.,
Prinz, M., 2013. A new type of microglia gene targeting shows TAK1 to be
pivotal in CNS autoimmune inflammation. Nat. Neurosci. 16, 1618e1626.
Gonzalez-Scarano, F., Baltuch, G., 1999. Microglia as mediators of inflammatory and
degenerative diseases. Annu. Rev. Neurosci. 22, 219e240.
Gordon, S., 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3,
23e35.
Gordon, S., Martinez, F.O., 2010. Alternative activation of macrophages: mechanism
and functions. Immunity 32, 593e604.
Gordon, W.C., Bazan, N.G., 1990. Docosahexaenoic acid utilization during rod
photoreceptor cell renewal. J. Neurosci. 10, 2190e2202.
Gorovits, R., Avidan, N., Avisar, N., Shaked, I., Vardimon, L., 1997. Glutamine syn-
thetase protects against neuronal degeneration in injured retinal tissue. Proc.
Natl. Acad. Sci. U. S. A. 94, 7024e7029.
Grigsby, J.G., Cardona, S.M., Pouw, C.E., Muniz, A., Mendiola, A.S., Tsin, A.T.,
Allen, D.M., Cardona, A.E., 2014. The role of microglia in diabetic retinopathy.
J. Ophthalmol. 2014, 705783.
Grosche, A., Pannicke, T., Chen, J., Wiedemann, P., Reichenbach, A., Bringmann, A.,
2013. Disruption of endogenous purinergic signaling inhibits vascular endo-
thelial growth factor- and glutamate-induced osmotic volume regulation of
Müller glial cells in knockout mice. Ophthalmic. Res. 50, 209e214.
Gu, S.M., Thompson, D.A., Srikumari, C.R., Lorenz, B., Finckh, U., Nicoletti, A.,
Murthy, K.R., Rathmann, M., Kumaramanickavel, G., Denton, M.J., Gal, A., 1997.
Mutations in RPE65 cause autosomal recessive childhood-onset severe retinal
dystrophy. Nat. Genet. 17, 194e197.
Guidry, C., Bradley, K.M., King, J.L., 2003. Tractional force generation by human
muller cells: growth factor responsiveness and integrin receptor involvement.
Invest. Ophthalmol. Vis. Sci. 44, 1355e1363.
Guvakova, M.A., 2007. Insulin-like growth factors control cell migration in health
and disease. Int. J. Biochem. Cell. Biol. 39, 890e909.
Hajrasouliha, A.R., Jiang, G., Lu, Q., Lu, H., Kaplan, H.J., Zhang, H.G., Shao, H., 2013.
Exosomes from retinal astrocytes contain antiangiogenic components that
inhibit laser-induced choroidal neovascularization. J. Biol. Chem. 288,
28058e28067.
Hall, D.E., Neugebauer, K.M., Reichardt, L.F., 1987. Embryonic neural retinal cell
35
response to extracellular matrix proteins: developmental changes and effects of
the cell substratum attachment antibody (CSAT). J. Cell. Biol. 104, 623e634.
Hama, K., Mizukawa, A., Kosaka, T., 1978. Fine structure of the Muller cell revealed
by high-voltage electron microscopy. Sens. Process. 2, 296e299.
Harada, C., Harada, T., Quah, H.M., Maekawa, F., Yoshida, K., Ohno, S., Wada, K.,
Parada, L.F., Tanaka, K., 2003. Potential role of glial cell line-derived neuro-
trophic factor receptors in Muller glial cells during light-induced retinal
degeneration. Neuroscience 122, 229e235.
Harada, C., Harada, T., Slusher, B.S., Yoshida, K., Matsuda, H., Wada, K., 2000. N-
acetylated-alpha-linked-acidic dipeptidase inhibitor has a neuroprotective ef-
fect on mouse retinal ganglion cells after pressure-induced ischemia. Neurosci.
Lett. 292, 134e136.
Harada, T., Harada, C., Kohsaka, S., Wada, E., Yoshida, K., Ohno, S., Mamada, H.,
Tanaka, K., Parada, L.F., Wada, K., 2002. Microglia-Müller glia cell interactions
control neurotrophic factor production during light-induced retinal degenera-
tion. J. Neurosci. 22, 9228e9236.
Harry, G.J., 2013. Microglia during development and aging. Pharmacol. Ther. 139,
313e326.
Hauck, S.M., Kinkl, N., Deeg, C.A., Swiatek-de Lange, M., Schoffmann, S., Ueffing, M.,
2006. GDNF family ligands trigger indirect neuroprotective signaling in retinal
glial cells. Mol. Cell. Biol. 26, 2746e2757.
Hauck, S.M., von Toerne, C., Ueffing, M., 2014. The neuroprotective potential of
retinal Müller glial cells. Adv. Exp. Med. Biol. 801, 381e387.
Hauswirth, W.W., Aleman, T.S., Kaushal, S., Cideciyan, A.V., Schwartz, S.B., Wang, L.,
Conlon, T.J., Boye, S.L., Flotte, T.R., Byrne, B.J., Jacobson, S.G., 2008. Treatment of
leber congenital amaurosis due to RPE65 mutations by ocular subretinal in-
jection of adeno-associated virus gene vector: short-term results of a phase I
trial. Hum. Gene Ther. 19, 979e990.
He, Y., Zhang, Y., Liu, X., Ghazaryan, E., Li, Y., Xie, J., Su, G., 2014. Recent advances of
stem cell therapy for retinitis pigmentosa. Int. J. Mol. Sci. 15, 14456e14474.
Heiduschka, P., Renninger, D., Fischer, D., Muller, A., Hofmeister, S., Schraermeyer, U.,
2013. Lens injury has a protective effect on photoreceptors in the RCS rat. ISRN
Ophthalmol. 2013, 814814.
Heins, N., Malatesta, P., Cecconi, F., Nakafuku, M., Tucker, K.L., Hack, M.A.,
Chapouton, P., Barde, Y.A., Gotz, M., 2002. Glial cells generate neurons: the role
of the transcription factor Pax6. Nat. Neurosci. 5, 308e315.
Hernandez, M., Pearce-Kelling, S.E., Rodriguez, F.D., Aguirre, G.D., Vecino, E., 2010.
Altered expression of retinal molecular markers in the canine RPE65 model of
Leber congenital amaurosis. Invest. Ophthalmol. Vis. Sci. 51, 6793e6802.
Hernandez, M., Rodriguez, F.D., Sharma, S.C., Vecino, E., 2009. Immunohistochem-
ical changes in rat retinas at various time periods of elevated intraocular
pressure. Mol. Vis. 15, 2696e2709.
Hernandez, M., Urcola, J.H., Vecino, E., 2008. Retinal ganglion cell neuroprotection
in a rat model of glaucoma following brimonidine, latanoprost or combined
treatments. Exp. Eye Res. 86, 798e806.
Hernandez, M.R., Pena, J.D., Selvidge, J.A., Salvador-Silva, M., Yang, P., 2000. Hy-
drostatic pressure stimulates synthesis of elastin in cultured optic nerve head
astrocytes. Glia 32, 122e136.
Higginson, J.R., Ruzafa, N., Rodríguez-Ferna
ndez, D., Vecino, E., 2013. Neuro-
protection of retinal ganglion cells by Müller glia and astrocytes. Poster. In: XI
European Meeting on Glial Cell Function in Health and Disease.
Hirrlinger, P.G., Wurm, A., Hirrlinger, J., Bringmann, A., Reichenbach, A., 2008. Os-
motic swelling characteristics of glial cells in the murine hippocampus, cere-
bellum, and retina in situ. J. Neurochem. 105, 1405e1417.
Hiscott, P., Waller, H.A., Grierson, I., Butler, M.G., Scott, D., 1992. Local production of
fibronectin by ectopic human retinal cells. Cell Tissue Res. 267, 185e192.
Hogan, M.J., Feeney, L., 1963. The ultrastructure of the retinal vessels. Iii. Vascular-
Glial relationships. J. Ultrastruct. Res. 49, 47e64.
Hogan, M.J., Moschini, G.B., Zardi, O., 1971. Effects of Toxoplasma gondii toxin on the
rabbit eye. Am. J. Ophthalmol. 72, 733e742.
Hollander, H., Makarov, F., Dreher, Z., van Driel, D., Chan-Ling, T.L., Stone, J., 1991.
Structure of the macroglia of the retina: sharing and division of labour between
astrocytes and Muller cells. J. Comp. Neurol. 313, 587e603.
Hollborn, M., Jahn, K., Limb, G.A., Kohen, L., Wiedemann, P., Bringmann, A., 2004.
Characterization of the basic fibroblast growth factor-evoked proliferation of
the human Muller cell line, MIO-M1. Graefes Arch. Clin. Exp. Ophthalmol. 242,
414e422.
Holley, J.E., Gveric, D., Whatmore, J.L., Gutowski, N.J., 2005. Tenascin C induces a
quiescent phenotype in cultured adult human astrocytes. Glia 52, 53e58.
Hosoya, K., Tachikawa, M., 2012. The inner blood-retinal barrier: molecular struc-
ture and transport biology. Adv. Exp. Med. Biol. 763, 85e104.
Hossmann, K.A., 1993. Ischemia-mediated neuronal injury. Resuscitation 26,
225e235.
Housley, G.D., Bringmann, A., Reichenbach, A., 2009. Purinergic signaling in special
senses. Trends Neurosci. 32, 128e141.
Howell, G.R., Libby, R.T., Marchant, J.K., Wilson, L.A., Cosma, I.M., Smith, R.S.,
Anderson, M.G., John, S.W., 2007. Absence of glaucoma in DBA/2J mice homo-
zygous for wild-type versions of Gpnmb and Tyrp1. BMC Genet. 8, 45.
Hu, P., Herrmann, R., Bednar, A., Saloupis, P., Dwyer, M.A., Yang, P., Qi, X.,
Thomas, R.S., Jaffe, G.J., Boulton, M.E., McDonnell, D.P., Malek, G., 2013. Aryl
hydrocarbon receptor deficiency causes dysregulated cellular matrix meta-
bolism and age-related macular degeneration-like pathology. Proc. Natl. Acad.
Sci. U. S. A. 110, E4069eE4078.
Hurley, J.B., Chertov, A.O., Lindsay, K., Giamarco, M., Cleghorn, W., Du, J.,
Brockerhoff, S., 2014. Vertebrate photoreceptors: functional molecular bases.
36 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
Energy Metabol. Vertebr. Retina.
Ibrahim, A.S., El-Remessy, A.B., Matragoon, S., Zhang, W., Patel, Y., Khan, S., Al-
Gayyar, M.M., El-Shishtawy, M.M., Liou, G.I., 2011. Retinal microglial activation
and inflammation induced by amadori-glycated albumin in a rat model of
diabetes. Diabetes 60, 1122e1133.
Inatani, M., Tanihara, H., Oohira, A., Honjo, M., Kido, N., Honda, Y., 2000. Upregu-
lated expression of neurocan, a nervous tissue specific proteoglycan, in tran-
sient retinal ischemia. Invest. Ophthalmol. Vis. Sci. 41, 2748e2754.
Jayaram, H., Jones, M.F., Eastlake, K., Cottrill, P.B., Becker, S., Wiseman, J., Khaw, P.T.,
Limb, G.A., 2014. Transplantation of photoreceptors derived from human Muller
glia restore rod function in the P23H rat. Stem Cells Transl. Med. 3, 323e333.
Jeon, C.J., Masland, R.H., 1993. Selective accumulation of diamidino yellow and
chromomycin A3 by retinal glial cells. J. Histochem. Cytochem. 41, 1651e1658.
Jerdan, J.A., Glaser, B.M., 1986. Retinal microvessel extracellular matrix: an immu-
nofluorescent study. Invest. Ophthalmol. Vis. Sci. 27, 194e203.
Jha, M.K., Seo, M., Kim, J.H., Kim, B.G., Cho, J.Y., Suk, K., 2013. The secretome
signature of reactive glial cells and its pathological implications. Biochim. Bio-
phys. Acta 1834, 2418e2428.
Jiang, B., Liou, G.I., Behzadian, M.A., Caldwell, R.B., 1994. Astrocytes modulate retinal
vasculogenesis: effects on fibronectin expression. J. Cell Sci. 107 (Pt 9),
2499e2508.
Johnson, E.C., Jia, L., Cepurna, W.O., Doser, T.A., Morrison, J.C., 2007. Global changes
in optic nerve head gene expression after exposure to elevated intraocular
pressure in a rat glaucoma model. Invest. Ophthalmol. Vis. Sci. 48, 3161e3177.
Jonas, R.A., Yuan, T.F., Liang, Y.X., Jonas, J.B., Tay, D.K., Ellis-Behnke, R.G., 2012. The
spider effect: morphological and orienting classification of microglia in
response to stimuli in vivo. PLoS One 7, e30763.
Kang, J., Jiang, L., Goldman, S.A., Nedergaard, M., 1998. Astrocyte-mediated poten-
tiation of inhibitory synaptic transmission. Nat. Neurosci. 1, 683e692.
Kang, X., Yu, J., Wei, Y., Zhao, P., 2013. The effect of A2A receptor sntagonist on the
mRNA expression of Kir 2.1 and Kir 4.1 channels in rat retinal Müller cells under
hypoxic conditions in vitro. Adv. Clin. Exp. Med. 22, 825e829.
Karlstetter, M., Ebert, S., Langmann, T., 2010. Microglia in the healthy and degen-
erating retina: insights from novel mouse models. Immunobiology 215,
685e691.
Karlstetter, M., Langmann, T., 2014. Microglia in the aging retina. Adv. Exp. Med.
Biol. 801, 207e212.
Karschin, A., Wassle, H., Schnitzer, J., 1986. Shape and distribution of astrocytes in
the cat retina. Invest. Ophthalmol. Vis. Sci. 27, 828e831.
Katsman, D., Stackpole, E.J., Domin, D.R., Farber, D.B., 2012. Embryonic stem cell-
derived microvesicles induce gene expression changes in Muller cells of the
retina. PLoS One 7, e50417.
Kawikova, I., Askenase, P.W., 2014. Diagnostic and therapeutic potentials of exo-
somes in CNS diseases. Brain Res.
Keenan, T.D., Clark, S.J., Unwin, R.D., Ridge, L.A., Day, A.J., Bishop, P.N., 2012. Mapping
the differential distribution of proteoglycan core proteins in the adult human
retina, choroid, and sclera. Invest. Ophthalmol. Vis. Sci. 53, 7528e7538.
Kettenmann, H., Hanisch, U.K., Noda, M., Verkhratsky, A., 2011. Physiology of
microglia. Physiol. Rev. 91, 461e553.
Kettenmann, H., Kirchhoff, F., Verkhratsky, A., 2013. Microglia: new roles for the
synaptic stripper. Neuron 77, 10e18.
Kettenmann, H., Verkhratsky, A., 2008. Neuroglia: the 150 years after. Trends
Neurosci. 31, 653e659.
Kezic, J.M., Chen, X., Rakoczy, E.P., McMenamin, P.G., 2013. The effects of age and
Cx3cr1 deficiency on retinal microglia in the Ins2(Akita) diabetic mouse. Invest.
Ophthalmol. Vis. Sci. 54, 854e863.
Kierdorf, K., Erny, D., Goldmann, T., Sander, V., Schulz, C., Perdiguero, E.G.,
Wieghofer, P., Heinrich, A., Riemke, P., Holscher, C., Muller, D.N., Luckow, B.,
Brocker, T., Debowski, K., Fritz, G., Opdenakker, G., Diefenbach, A., Biber, K.,
Heikenwalder, M., Geissmann, F., Rosenbauer, F., Prinz, M., 2013. Microglia
emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent path-
ways. Nat. Neurosci. 16, 273e280.
Kim, I.B., Kim, K.Y., Joo, C.K., Lee, M.Y., Oh, S.J., Chung, J.W., Chun, M.H., 1998. Re-
action of Muller cells after increased intraocular pressure in the rat retina. Exp.
Brain Res. 121, 419e424.
Kim, J.H., Kim, J.H., Park, J.A., Lee, S.W., Kim, W.J., Yu, Y.S., Kim, K.W., 2006. Blood-
neural barrier: intercellular communication at glio-vascular interface.
J. Biochem. Mol. Biol. 39, 339e345.
Kim, S.Y., Yang, H.J., Chang, Y.S., Kim, J.W., Brooks, M., Chew, E.Y., Wong, W.T.,
Fariss, R.N., Rachel, R.A., Cogliati, T., Qian, H., Swaroop, A., 2014. Deletion of aryl
hydrocarbon receptor AHR in mice leads to subretinal accumulation of micro-
glia and RPE atrophy. Invest. Ophthalmol. Vis. Sci. 55, 6031e6040.
Kingma, P.B., Bok, D., Ong, D.E., 1998. Bovine epidermal fatty acid-binding protein:
determination of ligand specificity and cellular localization in retina and testis.
Biochemistry 37, 3250e3257.
Kinkl, N., Ruiz, J., Vecino, E., Frasson, M., Sahel, J., Hicks, D., 2003. Possible
involvement of a fibroblast growth factor 9 (FGF9)-FGF receptor-3-mediated
pathway in adult pig retinal ganglion cell survival in vitro. Mol. Cell Neurosci.
23, 39e53.
Kinoshita, J.H., 1986. Aldose reductase in the diabetic eye. XLIII Edward Jackson
memorial lecture. Am. J. Ophthalmol. 102, 685e692.
Kitahara, A., Toyota, T., Kakizaki, M., Goto, Y., 1978. Activities of hepatic enzymes in
spontaneous diabetes rats produced by selective breeding of normal Wistar
rats. Tohoku J. Exp. Med. 126, 7e11.
Kitano, S., Morgan, J., Caprioli, J., 1996. Hypoxic and excitotoxic damage to cultured
rat retinal ganglion cells. Exp. Eye Res. 63, 105e112.
Kofuji, P., Ceelen, P., Zahs, K.R., Surbeck, L.W., Lester, H.A., Newman, E.A., 2000.
Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 sub-
unit) in mice: phenotypic impact in retina. J. Neurosci. 20, 5733e5740.
Kohno, H., Sakai, T., Kitahara, K., 2006. Induction of nestin, Ki-67, and cyclin D1
expression in Muller cells after laser injury in adult rat retina. Graefes Arch.
Clin. Exp. Ophthalmol. 244, 90e95.
Kohno, T., Sorgente, N., Ishibashi, T., Goodnight, R., Ryan, S.J., 1987. Immunofluo-
rescent studies of fibronectin and laminin in the human eye. Invest. Oph-
thalmol. Vis. Sci. 28, 506e514.
Kokaia, Z., Airaksinen, M.S., Nanobashvili, A., Larsson, E., Kujamaki, E., Lindvall, O.,
Saarma, M., 1999. GDNF family ligands and receptors are differentially regulated
after brain insults in the rat. Eur. J. Neurosci. 11, 1202e1216.
Koke, J.R., Mosier, A.L., Garcia, D.M., 2010. Intermediate filaments of zebrafish retinal
and optic nerve astrocytes and Muller glia: differential distribution of cyto-
keratin and GFAP. BMC Res. Notes 3, 50.
Krady, J.K., Basu, A., Allen, C.M., Xu, Y., LaNoue, K.F., Gardner, T.W., Levison, S.W.,
2005. Minocycline reduces proinflammatory cytokine expression, microglial
activation, and caspase-3 activation in a rodent model of diabetic retinopathy.
Diabetes 54, 1559e1565.
Kuehn, M.H., Kim, C.Y., Ostojic, J., Bellin, M., Alward, W.L., Stone, E.M.,
Sakaguchi, D.S., Grozdanic, S.D., Kwon, Y.H., 2006. Retinal synthesis and depo-
sition of complement components induced by ocular hypertension. Exp. Eye.
Res. 83, 620e628.
Kumar, A., Pandey, R.K., Miller, L.J., Singh, P.K., Kanwar, M., 2013. Muller glia in
retinal innate immunity: a perspective on their roles in endophthalmitis. Crit.
Rev. Immunol. 33, 119e135.
Labin, A.M., Ribak, E.N., 2010. Retinal glial cells enhance human vision acuity. Phys.
Rev. Lett. 104, 158102.
Lange, J., Yafai, Y., Noack, A., Yang, X.M., Munk, A.B., Krohn, S., Iandiev, I.,
Wiedemann, P., Reichenbach, A., Eichler, W., 2012. The axon guidance molecule
Netrin-4 is expressed by Muller cells and contributes to angiogenesis in the
retina. Glia 60, 1567e1578.
Langmann, T., 2007. Microglia activation in retinal degeneration. J. Leukoc. Biol. 81,
1345e1351.
Larger, E., Becourt, C., Bach, J.F., Boitard, C., 1995. Pancreatic islet beta cells drive T
cell-immune responses in the nonobese diabetic mouse model. J. Exp. Med. 181,
1635e1642.
Laties, A.M., 1983. New light on the Muller cell. Trans. Ophthalmol. Soc. U. K. 103 (Pt
4), 380e384.
Lawrence, J.M., Singhal, S., Bhatia, B., Keegan, D.J., Reh, T.A., Luthert, P.J., Khaw, P.T.,
Limb, G.A., 2007. MIO-M1 cells and similar muller glial cell lines derived from
adult human retina exhibit neural stem cell characteristics. Stem Cells 25,
2033e2043.
Leveillard, T., Fridlich, R., Clerin, E., Ait-Ali, N., Millet-Puel, G., Jaillard, C., Yang, Y.,
Zack, D., van-Dorsselaer, A., Sahel, J.A., 2014. Therapeutic strategy for handling
inherited retinal degenerations in a gene-independent manner using rod-
derived cone viability factors. C. R. Biol. 337, 207e213.
Lewis, G.P., Fisher, S.K., 2003. Up-regulation of glial fibrillary acidic protein in
response to retinal injury: its potential role in glial remodeling and a com-
parison to vimentin expression. Int. Rev. Cytol. 230, 263e290.
Lewis, G.P., Guerin, C.J., Anderson, D.H., Matsumoto, B., Fisher, S.K., 1994. Rapid
changes in the expression of glial cell proteins caused by experimental retinal
detachment. Am. J. Ophthalmol. 118, 368e376.
Li, J., Zhao, S.Z., Wang, P.P., Yu, S.P., Zheng, Z., Xu, X., 2012. Calcium mediates high
glucose-induced HIF-1a and VEGF expression in cultured rat retinal Müller cells
through CaMKII-CREB pathway. Acta Pharmacol. Sin. 33, 1030e1036.
Libby, R.T., Champliaud, M.F., Claudepierre, T., Xu, Y., Gibbons, E.P., Koch, M.,
Burgeson, R.E., Hunter, D.D., Brunken, W.J., 2000. Laminin expression in adult
and developing retinae: evidence of two novel CNS laminins. J. Neurosci. 20,
6517e6528.
Light, J.G., Fransen, J.W., Adekunle, A.N., Adkins, A., Pangeni, G., Loudin, J.,
Mathieson, K., Palanker, D.V., McCall, M.A., Pardue, M.T., 2014. Inner retinal
preservation in rat models of retinal degeneration implanted with subretinal
photovoltaic arrays. Exp. Eye Res. 128C, 34e42.
Limb, G.A., Salt, T.E., Munro, P.M., Moss, S.E., Khaw, P.T., 2002. In vitro character-
ization of a spontaneously immortalized human Muller cell line (MIO-M1).
Invest. Ophthalmol. Vis. Sci. 43, 864e869.
Lindqvist, N., Liu, Q., Zajadacz, J., Franze, K., Reichenbach, A., 2010. Retinal glial
(Müller) cells: sensing and responding to tissue stretch. Invest. Ophthalmol. Vis.
Sci. 51, 1683e1690.
Linser, P., Moscona, A.A., 1981. Carbonic anhydrase C in the neural retina: transition
from generalized to glia-specific cell localization during embryonic develop-
ment. Proc. Natl. Acad. Sci. U. S. A. 78, 7190e7194.
Liu, B., Hunter, D.J., Smith, A.A., Chen, S., Helms, J.A., 2014a. The capacity of neural
crest-derived stem cells for ocular repair. Birth Defects Res. Part C Embryo
Today Rev. 102, 299e308.
Liu, Q., Londraville, R.L., Azodi, E., Babb, S.G., Chiappini-Williamson, C., Marrs, J.A.,
Raymond, P.A., 2002. Up-regulation of cadherin-2 and cadherin-4 in regener-
ating visual structures of adult zebrafish. Exp. Neurol. 177, 396e406.
Liu, R.T., Gao, J., Cao, S., Sandhu, N., Cui, J.Z., Chou, C.L., Fang, E., Matsubara, J.A., 2013.
Inflammatory mediators induced by amyloid-beta in the retina and RPE in vivo:
implications for inflammasome activation in age-related macular degeneration.
Invest. Ophthalmol. Vis. Sci. 54, 2225e2237.
Liu, X., Ye, F., Xiong, H., Hu, D., Limb, G.A., Xie, T., Peng, L., Yang, W., Sun, Y., Zhou, M.,
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
Song, E., Zhang, D.Y., 2014b. IL-1beta upregulates IL-8 production in human
Muller Cells through activation of the p38 MAPK and ERK1/2 signaling path-
ways. Inflammation 37, 1486e1495.
Liu, X., Ye, F., Xiong, H., Hu, D.-N., Limb, G.A., Xie, T., Peng, L., Zhang, P., Wei, Y.,
Zhang, W., Wang, J., Wu, H., Leei, P., Songa, E., Zhangb, D.Y., 2014c. IL-1b induces
IL-6 production in retinal Müller cells predominantly through the activation of
P38 MAPK/NF-kB signaling pathway. Exp. Cell Res.
Ljubimov, A.V., Burgeson, R.E., Butkowski, R.J., Couchman, J.R., Zardi, L., Ninomiya, Y.,
Sado, Y., Huang, Z.S., Nesburn, A.B., Kenney, M.C., 1996. Basement membrane
abnormalities in human eyes with diabetic retinopathy. J. Histochem. Cyto-
chem. 44, 1469e1479.
Lopez-Colome, A.M., Martinez-Lozada, Z., Guillem, A.M., Lopez, E., Ortega, A., 2012.
Glutamate transporter-dependent mTOR phosphorylation in Muller glia cells.
ASN Neuro 4, e00095.
Lorber, B., Berry, M., Douglas, M.R., Nakazawa, T., Logan, A., 2009. Activated retinal
glia promote neurite outgrowth of retinal ganglion cells via apolipoprotein E.
J. Neurosci. Res. 87, 2645e2652.
Lorber, B., Berry, M., Logan, A., 2008. Different factors promote axonal regeneration
of adult rat retinal ganglion cells after lens injury and intravitreal peripheral
nerve grafting. J. Neurosci. Res. 86, 894e903.
Lorber, B., Berry, M., Logan, A., Tonge, D., 2002. Effect of lens lesion on neurite
outgrowth of retinal ganglion cells in vitro. Mol. Cell. Neurosci. 21, 301e311.
Lorber, B., Guidi, A., Fawcett, J.W., Martin, K.R., 2012. Activated retinal glia mediated
axon regeneration in experimental glaucoma. Neurobiol. Dis. 45, 243e252.
Lu, Y.-B., Pannicke, T., Wei, E.-Q., Bringmann, A., Wiedemann, P., Habermann, G.,
Buse, E., Ka €s, J.A., Reichenbach, A., 2013. Biomechanical properties of retinal
glial cells: comparative and developmental data. Exp. Eye Res. 113, 60e65.
Lu, Y.B., Franze, K., Seifert, G., Steinhauser, C., Kirchhoff, F., Wolburg, H., Guck, J.,
Janmey, P., Wei, E.Q., Kas, J., Reichenbach, A., 2006. Viscoelastic properties of
individual glial cells and neurons in the CNS. Proc. Natl. Acad. Sci. U. S. A. 103,
17759e17764.
Lu, Y.B., Iandiev, I., Hollborn, M., Korber, N., Ulbricht, E., Hirrlinger, P.G., Pannicke, T.,
Wei, E.Q., Bringmann, A., Wolburg, H., Wilhelmsson, U., Pekny, M.,
Wiedemann, P., Reichenbach, A., Kas, J.A., 2011. Reactive glial cells: increased
stiffness correlates with increased intermediate filament expression. FASEB J.
25, 624e631.
Luna, G., Lewis, G.P., Banna, C.D., Skalli, O., Fisher, S.K., 2010. Expression profiles of
nestin and synemin in reactive astrocytes and Muller cells following retinal
injury: a comparison with glial fibrillar acidic protein and vimentin. Mol. Vis. 16,
2511e2523.
Lundkvis, A., Reichenbach, A., Betsholtz, C., Carmeliet, P., Wolburg, H., Pekny, M.,
2004. Under stress, the absence of intermediate filaments from Müller cells in
the retina has structural and functional consequences. J. Cell. Sci. 117,
3481e3488.
Luo, C., Chen, M., Xu, H., 2011. Complement gene expression and regulation in
mouse retina and retinal pigment epithelium/choroid. Mol. Vis. 17, 1588e1597.
Lye-Barthel, M., Sun, D., Jakobs, T.C., 2013. Morphology of astrocytes in a glau-
comatous optic nerve. Invest. Ophthalmol. Vis. Sci. 54, 909e917.
Ma, W., Zhao, L., Fontainhas, A.M., Fariss, R.N., Wong, W.T., 2009. Microglia in the
mouse retina alter the structure and function of retinal pigmented epithelial
cells: a potential cellular interaction relevant to AMD. PLoS One 4, e7945.
Ma, W., Zhao, L., Wong, W.T., 2012. Microglia in the outer retina and their relevance
to pathogenesis of age-related macular degeneration. Adv. Exp. Med. Biol. 723,
37e42.
Ma, Y., Qu, Y., Fei, Z., 2011. Vascular endothelial growth factor in cerebral ischemia.
J. Neurosci. Res. 89, 969e978.
Mansford, K.R., Opie, L., 1968. Comparison of metabolic abnormalities in diabetes
mellitus induced by streptozotocin or by alloxan. Lancet 1, 670e671.
Marc, R.E., 1992. Structural organization of GABAergic circuitry in ectotherm retinas.
Prog. Brain Res. 90, 61e92.
Mastellos, D.C., 2014. Complement emerges as a masterful regulator of CNS ho-
meostasis, neural synaptic plasticity and cognitive function. Exp. Neurol. 261,
469e474.
Matteucci, A., Gaddini, L., Villa, M., Varano, M., Parravano, M.C., Monteleone, V.,
Cavallo, F., Leo, L., Mallozzi, C., Malchiodi-Albedi, F., Pricci, F., 2014. Neuro-
protection by rat Müller glia against high glucose-induced neurodegeneration
through a mechanism involving ERK1/2 activation. Exp. Eye Res. 125, 20e29.
Matyash, V., Kettenmann, H., 2010. Heterogeneity in astrocyte morphology and
physiology. Brain Res. Rev. 63, 2e10.
Mauch, D.H., Nagler, K., Schumacher, S., Goritz, C., Muller, E.C., Otto, A.,
Pfrieger, F.W., 2001. CNS synaptogenesis promoted by glia-derived cholesterol.
Science 294, 1354e1357.
Medzhitov, R., 2008. Origin and physiological roles of inflammation. Nature 454,
428e435.
Medzhitov, R., 2010. Inflammation 2010: new adventures of an old flame. Cell 140,
771e776.
Mesquida, M., Leszczynska, A., Llorenc, V., Adan, A., 2014. Interleukin-6 blockade in
ocular inflammatory diseases. Clin. Exp. Immunol. 176, 301e309.
Mi, X.S., Yuan, T.F., So, K.F., 2014. The current research status of normal tension
glaucoma. Clin. Interv. Aging 9, 1563e1571.
Miyamoto, A., Wake, H., Moorhouse, A.J., Nabekura, J., 2013. Microglia and synapse
interactions: fine tuning neural circuits and candidate molecules. Front. Cell.
Neurosci. 7, 70.
Mizutani, M., Pino, P.A., Saederup, N., Charo, I.F., Ransohoff, R.M., Cardona, A.E.,
2012. The fractalkine receptor but not CCR2 is present on microglia from
37
embryonic development throughout adulthood. J. Immunol. 188, 29e36.
Morcos, Y., Chan-Ling, T., 2000. Concentration of astrocytic filaments at the retinal
optic nerve junction is coincident with the absence of intra-retinal myelination:
comparative and developmental evidence. J. Neurocytol. 29, 665e678.
Morishita, S., Oku, H., Horie, T., Tonari, M., Kida, T., Okubo, A., Sugiyama, T., Takai, S.,
Hara, H., Ikeda, T., 2014. Systemic simvastatin rescues retinal ganglion cells from
optic nerve injury possibly through suppression of astroglial NF-kappaB acti-
vation. PLoS One 9, e84387.
Morissette, N., Carbonetto, S., 1995. Laminin alpha 2 chain (M chain) is found within
the pathway of avian and murine retinal projections. J. Neurosci. 15,
8067e8082.
Morrison, J.C., 2006. Integrins in the optic nerve head: potential roles in glau-
comatous optic neuropathy (an American Ophthalmological Society thesis).
Trans. Am. Ophthalmol. Soc. 104, 453e477.
Moscona, A.A., Fox, L., Smith, J., Degenstein, L., 1985. Antiserum to lens antigens
immunostains Muller glia cells in the neural retina. Proc. Natl. Acad. Sci. U. S. A.
82, 5570e5573.
Muller, A., Hauk, T.G., Fischer, D., 2007. Astrocyte-derived CNTF switches mature
RGCs to a regenerative state following inflammatory stimulation. Brain 130,
3308e3320.
Nagelhus, E.A., Ottersen, O.P., 2013. Physiological roles of aquaporin-4 in brain.
Physiol. Rev. 93, 1543e1562.
Nahirnyj, A., Livne-Bar, I., Guo, X., Sivak, J.M., 2013. ROS detoxification and proin-
flammatory cytokines are linked by p38 MAPK signaling in a model of mature
astrocyte activation. PLoS One 8, e83049.
Nakazawa, T., Matsubara, A., Noda, K., Hisatomi, T., She, H., Skondra, D., Miyahara, S.,
Sobrin, L., Thomas, K.L., Chen, D.F., Grosskreutz, C.L., Hafezi-Moghadam, A.,
Miller, J.W., 2006. Characterization of cytokine responses to retinal detachment
in rats. Mol. Vis. 12, 867e878.
Neugebauer, K.M., Tomaselli, K.J., Lilien, J., Reichardt, L.F., 1988. N-cadherin, NCAM,
and integrins promote retinal neurite outgrowth on astrocytes in vitro. J. Cell
Biol. 107, 1177e1187.
Newman, E., Reichenbach, A., 1996. The Muller cell: a functional element of the
retina. Trends Neurosci. 19, 307e312.
Newman, E.A., 2003. New roles for astrocytes: regulation of synaptic transmission.
Trends Neurosci. 26, 536e542.
Newman, E.A., Frambach, D.A., Odette, L.L., 1984. Control of extracellular potassium
levels by retinal glial cell Kþ siphoning. Science 225, 1174e1175.
Ng, S.K., Wood, J.P., Chidlow, G., Han, G., Kittipassorn, T., Peet, D.J., Casson, R.J., 2014.
Cancer-like metabolism of the mammalian retina. Clin. Exp. Ophthalmol.
Nguyen, J.V., Soto, I., Kim, K.Y., Bushong, E.A., Oglesby, E., Valiente-Soriano, F.J.,
Yang, Z., Davis, C.H., Bedont, J.L., Son, J.L., Wei, J.O., Buchman, V.L., Zack, D.J.,
Vidal-Sanz, M., Ellisman, M.H., Marsh-Armstrong, N., 2011. Myelination transi-
tion zone astrocytes are constitutively phagocytic and have synuclein depen-
dent reactivity in glaucoma. Proc. Natl. Acad. Sci. U. S. A. 108, 1176e1181.
Noailles, A., Fernandez-Sanchez, L., Lax, P., Cuenca, N., 2014. Microglia activation in a
model of retinal degeneration and TUDCA neuroprotective effects.
J. Neuroinflammation 11, 186.
Oberheim, N.A., Takano, T., Han, X., He, W., Lin, J.H., Wang, F., Xu, Q., Wyatt, J.D.,
Pilcher, W., Ojemann, J.G., Ransom, B.R., Goldman, S.A., Nedergaard, M., 2009.
Uniquely hominid features of adult human astrocytes. J. Neurosci. 29,
3276e3287.
Ogden, T.E., 1978. Nerve fiber layer astrocytes of the primate retina: morphology,
distribution, and density. Invest. Ophthalmol. Vis. Sci. 17, 499e510.
Ola, M.S., Nawaz, M.I., Siddiquei, M.M., Al-Amro, S., Abu El-Asrar, A.M., 2012. Recent
advances in understanding the biochemical and molecular mechanism of dia-
betic retinopathy. J. Diabetes Complicat. 26, 56e64.
Oosthuyse, B., Moons, L., Storkebaum, E., Beck, H., Nuyens, D., Brusselmans, K., Van
Dorpe, J., Hellings, P., Gorselink, M., Heymans, S., Theilmeier, G., Dewerchin, M.,
Laudenbach, V., Vermylen, P., Raat, H., Acker, T., Vleminckx, V., Van Den
Bosch, L., Cashman, N., Fujisawa, H., Drost, M.R., Sciot, R., Bruyninckx, F.,
Hicklin, D.J., Ince, C., Gressens, P., Lupu, F., Plate, K.H., Robberecht, W.,
Herbert, J.M., Collen, D., Carmeliet, P., 2001. Deletion of the hypoxia-response
element in the vascular endothelial growth factor promoter causes motor
neuron degeneration. Nat. Genet. 28, 131e138.
Ooto, S., Akagi, T., Kageyama, R., Akita, J., Mandai, M., Honda, Y., Takahashi, M., 2004.
Potential for neural regeneration after neurotoxic injury in the adult mamma-
lian retina. Proc. Natl. Acad. Sci. U. S. A. 101, 13654e13659.
Overby, D.R., Bertrand, J., Tektas, O.Y., Boussommier-Calleja, A., Schicht, M.,
Ethier, C.R., Woodward, D.F., Stamer, W.D., Lutjen-Drecoll, E., 2014. Ultrastruc-
tural changes associated with dexamethasone-induced ocular hypertension in
mice. Invest. Ophthalmol. Vis. Sci. 55, 4922e4933.
Ozaki, H., Seo, M.S., Ozaki, K., Yamada, H., Yamada, E., Okamoto, N., Hofmann, F.,
Wood, J.M., Campochiaro, P.A., 2000. Blockade of vascular endothelial cell
growth factor receptor signaling is sufficient to completely prevent retinal
neovascularization. Am. J. Pathol. 156, 697e707.
Pannicke, T., Wurm, A., Iandiev, I., Hollborn, M., Linnertz, R., Binder, D.K., Kohen, L.,
Wiedemann, P., Steinha €user, C., Reichenbach, A., Bringmann, A., 2010. Deletion
of aquaporin-4 renders retinal glial cells more susceptible to osmotic stress.
J. Neurosci. Res. 88, 2877e2888.
Paolicelli, R.C., Bolasco, G., Pagani, F., Maggi, L., Scianni, M., Panzanelli, P.,
Giustetto, M., Ferreira, T.A., Guiducci, E., Dumas, L., Ragozzino, D., Gross, C.T.,
2011. Synaptic pruning by microglia is necessary for normal brain development.
Science 333, 1456e1458.
Park, S., Lee, Y.J., 2013. Nano-mechanical compliance of Müller cells investigated by
38 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
atomic force microscopy. Int. J. Biol. Sci. 9, 702e706.
Parysek, L.M., del Cerro, M., Olmsted, J.B., 1985. Microtubule-associated protein 4
antibody: a new marker for astroglia and oligodendroglia. Neuroscience 15,
869e875.
Pasti, L., Volterra, A., Pozzan, T., Carmignoto, G., 1997. Intracellular calcium oscilla-
tions in astrocytes: a highly plastic, bidirectional form of communication be-
tween neurons and astrocytes in situ. J. Neurosci. 17, 7817e7830.
Paulson, O.B., Newman, E.A., 1987. Does the release of potassium from astrocyte
endfeet regulate cerebral blood flow? Science 237, 896e898.
Pekny, M., 2001. Astrocytic intermediate filaments: lessons from GFAP and vimentin
knock-out mice. Prog. Brain Res. 132, 23e30.
Pekny, M., Nilsson, M., 2005. Astrocyte activation and reactive gliosis. Glia 50,
427e434.
Pekny, M., Pekna, M., 2014. Astrocyte reactivity and reactive astrogliosis: costs and
benefits. Physiol. Rev. 94, 1077e1098.
Pekny, M., Wilhelmsson, U., Pekna, M., 2014. The dual role of astrocyte activation
and reactive gliosis. Neurosci. Lett. 565, 30e38.
Pena, J.D., Varela, H.J., Ricard, C.S., Hernandez, M.R., 1999. Enhanced tenascin
expression associated with reactive astrocytes in human optic nerve heads with
primary open angle glaucoma. Exp. Eye Res. 68, 29e40.
Peng, B., Xiao, J., Wang, K., So, K.F., Tipoe, G.L., Lin, B., 2014. Suppression of microglial
activation is neuroprotective in a mouse model of human retinitis pigmentosa.
J. Neurosci. 34, 8139e8150.
Perez-Alvarez, M.J., Isiegas, C., Santano, C., Salazar, J.J., Ramirez, A.I., Trivino, A.,
Ramirez, J.M., Albar, J.P., de la Rosa, E.J., Prada, C., 2008. Vimentin isoform
expression in the human retina characterized with the monoclonal antibody
3CB2. J. Neurosci. Res. 86, 1871e1883.
Pfrieger, F.W., Barres, B.A., 1996. New views on synapse-glia interactions. Curr. Opin.
Neurobiol. 6, 615e621.
Politi, L.E., Rotstein, N.P., Carri, N.G., 2001. Effect of GDNF on neuroblast proliferation
and photoreceptor survival: additive protection with docosahexaenoic acid.
Invest. Ophthalmol. Vis. Sci. 42, 3008e3015.
Pollak, J., Wilken, M.S., Ueki, Y., Cox, K.E., Sullivan, J.M., Taylor, R.J., Levine, E.M.,
Reh, T.A., 2013. ASCL1 reprograms mouse Müller glia into neurogenic retinal
progenitors. Development 140, 2619e2631.
Ponsioen, T.L., van der Worp, R.J., van Luyn, M.J., Hooymans, J.M., Los, L.I., 2005.
Packages of vitreous collagen (type II) in the human retina: an indication of
postnatal collagen turnover? Exp. Eye Res. 80, 643e650.
Ponsioen, T.L., van Luyn, M.J., van der Worp, R.J., Pas, H.H., Hooymans, J.M., Los, L.I.,
2008. Human retinal Muller cells synthesize collagens of the vitreous and vit-
reoretinal interface in vitro. Mol. Vis. 14, 652e660.
Porter, J.T., McCarthy, K.D., 1997. Astrocytic neurotransmitter receptors in situ and
in vivo. Prog. Neurobiol. 51, 439e455.
Prada, I., Furlan, R., Matteoli, M., Verderio, C., 2013. Classical and unconventional
pathways of vesicular release in microglia. Glia 61, 1003e1017.
Prinz, M., Priller, J., 2014. Microglia and brain macrophages in the molecular age:
from origin to neuropsychiatric disease. Nat. Rev. Neurosci. 15, 300e312.
Prinz, M., Priller, J., Sisodia, S.S., Ransohoff, R.M., 2011. Heterogeneity of CNS myeloid
cells and their roles in neurodegeneration. Nat. Neurosci. 14, 1227e1235.
Qin, Y., Ren, H., Hoffman, M.R., Fan, J., Zhang, M., Xu, G., 2012. Aquaporin changes
during diabetic retinopathy in rats are accelerated by systemic hypertension
and are linked to the renin-angiotensin system. Invest. Ophthalmol. Vis. Sci. 53,
3047e3053.
Quigley, H.A., 2011. Glaucoma. Lancet 377, 1367e1377.
Ramirez, A.I., Salazar, J.J., de Hoz, R., Rojas, B., Gallego, B.I., Salinas-Navarro, M.,
Alarcon-Martinez, L., Ortin-Martinez, A., Aviles-Trigueros, M., Vidal-Sanz, M.,
Trivino, A., Ramirez, J.M., 2010. Quantification of the effect of different levels of
IOP in the astroglia of the rat retina ipsilateral and contralateral to experimental
glaucoma. Invest. Ophthalmol. Vis. Sci. 51, 5690e5696.
Ramirez, M., Lamas, M., 2009. NMDA receptor mediates proliferation and CREB
phosphorylation in postnatal Müller glia-derived retinal progenitors. Mol. Vis.
15, 713e721.
Ransohoff, R.M., Brown, M.A., 2012. Innate immunity in the central nervous system.
J. Clin. Invest. 122, 1164e1171.
Ransohoff, R.M., Perry, V.H., 2009. Microglial physiology: unique stimuli, specialized
responses. Annu. Rev. Immunol. 27, 119e145.
Reichenbach, A., Bringmann, A., 2010. Müller Cells in the Healthy and Diseased
Retina. Springer, New York.
Reichenbach, A., Bringmann, A., 2013. New Funct. Müller Cells Glia 61, 651e678.
Reichenbach, A., Bringmann, A., 2015. Retinal Glia. Morgan & Claypool Publishers.
Reichenbach, A., Hagen, E., Schippel, K., Bruckner, G., Reichelt, W., Leibnitz, L., 1988.
Cytotopographical specialization of enzymatically isolated rabbit retinal Muller
(glial) cells: structure, ultrastructure, and 3H-ouabain binding sites. Z. Mikrosk.
Anat. Forsch 102, 897e912.
Reichenbach, A., Robinson, S.R., 1995. Phylogenetic constraints on retinal organi-
zation and development. In: Osborne, N., Chader, G. (Eds.), Progress in Retinal
and Eye Research. Pergamon Press., Oxford.
Reichenbach, A., Schneider, H., Leibnitz, L., Reichelt, W., Schaaf, P., Schumann, R.,
1989. The structure of rabbit retinal Muller (glial) cells is adapted to the sur-
rounding retinal layers. Anat. Embryol. (Berl.) 180, 71e79.
Reichenbach, A., Siegel, A., Rickmann, M., Wolff, J.R., Noone, D., Robinson, S.R., 1995.
Distribution of Bergmann glial somata and processes: implications for function.
J. Hirnforsch. 36, 509e517.
Reichenbach, A., Stolzenburg, J.U., Eberhardt, W., Chao, T.I., Dettmer, D., Hertz, L.,
1993. What do retinal muller (glial) cells do for their neuronal 'small siblings'?
J. Chem. Neuroanat. 6, 201e213.
Reichenbach, A., Wurm, A., Pannicke, T., Landiev, I., Wiedemann, P., Bringmann, A.,
2007. Muller cells as players in retinal degeneration and edema. Graefe Arch.
Clin. Exp. Ophthalmol. 245, 627e636.
Reyes-Aguirre, L.I., Ferraro, S., Quintero, H., Sa
nchez-Serrano, S.L., Go
mez-
Montalvo, A., Lamas, M., 2013. Glutamate-induced epigenetic and morpholog-
ical changes allow rat Müller cell dedifferentiation but not further acquisition of
a photoreceptor phenotype. Neuroscience 254, 347e360.
Rezaie, P., Male, D., 1999. Colonisation of the developing human brain and spinal
cord by microglia: a review. Microsc. Res. Tech. 45, 359e382.
Rhee, K.D., Nusinowitz, S., Chao, K., Yu, F., Bok, D., Yang, X.J., 2013. CNTF-mediated
protection of photoreceptors requires initial activation of the cytokine receptor
gp130 in Muller glial cells. Proc. Natl. Acad. Sci. U. S. A. 110, E4520eE4529.
Rhee, K.D., Yang, X.J., 2010. Function and mechanism of CNTF/LIF signaling in ret-
inogenesis. Adv. Exp. Med. Biol. 664, 647e654.
Rieck, J., 2013. The pathogenesis of glaucoma in the interplay with the immune
system. Invest. Ophthalmol. Vis. Sci. 54, 2393e2409.
Riehl, R., Johnson, K., Bradley, R., Grunwald, G.B., Cornel, E., Lilienbaum, A., Holt, C.E.,
1996. Cadherin function is required for axon outgrowth in retinal ganglion cells
in vivo. Neuron 17, 837e848.
Roberge, F.G., Caspi, R.R., Chan, C.C., Kuwabara, T., Nussenblatt, R.B., 1985. Long-term
culture of Muller cells from adult rats in the presence of activated lymphocytes/
monocytes products. Curr. Eye Res. 4, 975e982.
Roberge, F.G., Caspi, R.R., Chan, C.C., Nussenblatt, R.B., 1991. Inhibition of T
lymphocyte proliferation by retinal glial Muller cells: reversal of inhibition by
glucocorticoids. J. Autoimmun. 4, 307e314.
Roesch, K., Jadhav, A.P., Trimarchi, J.M., Stadler, M.B., Roska, B., Sun, B.B., Cepko, C.L.,
2008. The transcriptome of retinal Muller glial cells. J. Comp. Neurol. 509,
225e238.
Roesch, K., Stadler, M.B., Cepko, C.L., 2012. Gene expression changes within Muller
glial cells in retinitis pigmentosa. Mol. Vis. 18, 1197e1214.
Rogers, R.S., Dharsee, M., Ackloo, S., Sivak, J.M., Flanagan, J.G., 2012. Proteomics
analyses of human optic nerve head astrocytes following biomechanical strain.
Mol. Cell. Proteomics 11 (M111), 012302.
Rohen, J.W., Futa, R., Lutjen-Drecoll, E., 1981. The fine structure of the cribriform
meshwork in normal and glaucomatous eyes as seen in tangential sections.
Invest. Ophthalmol. Vis. Sci. 21, 574e585.
Rojas, B., Gallego, B.I., Ramirez, A.I., Salazar, J.J., de Hoz, R., Valiente-Soriano, F.J.,
Aviles-Trigueros, M., Villegas-Perez, M.P., Vidal-Sanz, M., Trivino, A.,
Ramirez, J.M., 2014. Microglia in mouse retina contralateral to experimental
glaucoma exhibit multiple signs of activation in all retinal layers.
J. Neuroinflammation 11, 133e2094, 2011-2133.
Rowan, S., Cepko, C.L., 2004. Genetic analysis of the homeodomain transcription
factor Chx10 in the retina using a novel multifunctional BAC transgenic mouse
reporter. Dev. Biol. 271, 388e402.
Ruilin, Z., Kin-Sang, C., Dong Feng, C., Liu, Y., 2014. Ephrin-A2 and -A3 are negative
regulators of the regenerative potential of Müller cells. Chin. Med. J. 127,
3438e3442.
Ruiz-Ederra, J., Garcia, M., Hernandez, M., Urcola, H., Hernandez-Barbachano, E.,
Araiz, J., Vecino, E., 2005. The pig eye as a novel model of glaucoma. Exp. Eye
Res. 81, 561e569.
Ruiz-Ederra, J., Hitchcock, P.F., Vecino, E., 2003. Two classes of astrocytes in the
adult human and pig retina in terms of their expression of high affinity NGF
receptor (TrkA). Neurosci. Lett. 337, 127e130.
Rungger-Brandle, E., Messerli, J.M., Niemeyer, G., Eppenberger, H.M., 1993. Confocal
microscopy and computer-assisted image reconstruction of astrocytes in the
mammalian retina. Eur. J. Neurosci. 5, 1093e1106.
Ruzafa, N., Rey-Santano, C., Mielgo, V., Rodríguez-Fernandez, D., Vecino, E., 2014.
SIRCOVA-OFTARED-RIG Joined Congress Abstracts: effect of hypoxia in neonatal
pig retina and Superior Colliculus. Ophthalmic Res. 52, 175e197.
Ryskamp, D.A., Witkovsky, P., Barabas, P., Huang, W., Koehler, C., Akimov, N.P.,
Lee, S.H., Chauhan, S., Xing, W., Renteria, R.C., Liedtke, W., Krizaj, D., 2011. The
polymodal ion channel transient receptor potential vanilloid 4 modulates cal-
cium flux, spiking rate, and apoptosis of mouse retinal ganglion cells.
J. Neurosci. 31, 7089e7101.
Saari, J.C., 2000. Biochemistry of visual pigment regeneration: the Friedenwald
lecture. Invest. Ophthalmol. Vis. Sci. 41, 337e348.
Saederup, N., Cardona, A.E., Croft, K., Mizutani, M., Cotleur, A.C., Tsou, C.L.,
Ransohoff, R.M., Charo, I.F., 2010. Selective chemokine receptor usage by central
nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice.
PLoS One 5, e13693.
Salazar, J.J., Gallego-Pinazo, R., de Hoz, R., Pinazo-Duran, M.D., Rojas, B.,
Ramirez, A.I., Serrano, M., Ramirez, J.M., 2013. “Super p53” mice display retinal
astroglial changes. PLoS One 8, e65446.
Sanderson, J., Dartt, D.A., Trinkaus-Randall, V., Pintor, J., Civan, M.M., Delamere, N.A.,
Fletcher, E.L., Salt, T.E., Grosche, A., Mitchell, C.H., 2014. Purines in the eye:
recent evidence for the physiological and pathological role of purines in the
RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea
and lacrimal gland. Exp. Eye Res. 127, 270e279.
Santiago, A.R., Baptista, F.I., Santos, P.F., Cristovao, G., Ambrosio, A.F., Cunha, R.A.,
Gomes, C.A., 2014. Role of microglia adenosine A(2A) receptors in retinal and
brain neurodegenerative diseases. Mediat. Inflamm. 2014, 465694.
Santos-Bredariol, A.S., Belmonte, M.A., Kihara, A.H., Santos, M.F., Hamassaki, D.E.,
2006. Small GTP-binding protein RhoB is expressed in glial Muller cells in the
vertebrate retina. J. Comp. Neurol. 494, 976e985.
 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
Sarthy, P.V., Fu, M., 1990. Localization of laminin B1 mRNA in retinal ganglion cells
by in situ hybridization. J. Cell Biol. 110, 2099e2108.
Sarthy, V., Ripps, H., 2001. Role in retinal pathophysiology. In: Blakemore, C. (Ed.),
The Retinal Müller Cell. Structure and Function. Kluwer Academic/Plenum
Publishers, New York, pp. 181e215.
Schafer, D.P., Lehrman, E.K., Kautzman, A.G., Koyama, R., Mardinly, A.R., Yamasaki, R.,
Ransohoff, R.M., Greenberg, M.E., Barres, B.A., Stevens, B., 2012. Microglia sculpt
postnatal neural circuits in an activity and complement-dependent manner.
Neuron 74, 691e705.
Schafer, D.P., Lehrman, E.K., Stevens, B., 2013. The “quad-partite” synapse:
microglia-synapse interactions in the developing and mature CNS. Glia 61,
24e36.
Scheef, E., Wang, S., Sorenson, C.M., Sheibani, N., 2005. Isolation and characteriza-
tion of murine retinal astrocytes. Mol. Vis. 11, 613e624.
Schubert, H.D., 1989. Cystoid macular edema: the apparent role of mechanical
factors. Prog. Clin. Biol. Res. 312, 277e291.
Schulz, C., Gomez Perdiguero, E., Chorro, L., Szabo-Rogers, H., Cagnard, N.,
Kierdorf, K., Prinz, M., Wu, B., Jacobsen, S.E., Pollard, J.W., Frampton, J., Liu, K.J.,
Geissmann, F., 2012. A lineage of myeloid cells independent of Myb and he-
matopoietic stem cells. Science (New York, N. Y. 336, 86e90.
Sheppard, A.M., Onken, M.D., Rosen, G.D., Noakes, P.G., Dean, D.C., 1994. Expanding
roles for alpha 4 integrin and its ligands in development. Cell Adhes. Commun.
2, 27e43.
Sheridan, G.K., Murphy, K.J., 2013. Neuron-glia crosstalk in health and disease:
fractalkine and CX3CR1 take centre stage. Open Biol. 3, 130181.
Shin, E.S., Huang, Q., Gurel, Z., Sorenson, C.M., Sheibani, N., 2014. High glucose alters
retinal astrocytes phenotype through increased production of inflammatory
cytokines and oxidative stress. PLoS One 9, e103148.
Siddiqui, S., Horvat-Brocker, A., Faissner, A., 2008. The glia-derived extracellular
matrix glycoprotein tenascin-C promotes embryonic and postnatal retina axon
outgrowth via the alternatively spliced fibronectin type III domain TNfnD.
Neuron Glia Biol. 4, 271e283.
Siddiqui, S., Horvat-Broecker, A., Faissner, A., 2009. Comparative screening of glial
cell types reveals extracellular matrix that inhibits retinal axon growth in a
chondroitinase ABC-resistant fashion. Glia 57, 1420e1438.
Sidman, R.L., 1961. Histogenesis of mouse retina studied with thymidine-H3. In:
Smelser, G.K. (Ed.), The Structure of the Eye. Academic Press, New York and
London, pp. 487e506.
Singh, P.K., Shiha, M.J., Kumar, A., 2014. Antibacterial responses of retinal Müller
glia: production of antimicrobial peptides, oxidative burst and phagocytosis.
J. Neuroinflammation 11, 33.
Singhal, S., Bhatia, B., Jayaram, H., Becker, S., Jones, M.F., Cottrill, P.B., Khaw, P.T.,
Salt, T.E., Limb, G.A., 2012. Human Muller glia with stem cell characteristics
differentiate into retinal ganglion cell (RGC) precursors in vitro and partially
restore RGC function in vivo following transplantation. Stem Cells Transl. Med.
1, 188e199.
Skoff, R.P., 1990. Gliogenesis in rat optic nerve: astrocytes are generated in a single
wave before oligodendrocytes. Dev. Biol. 139, 149e168.
Smedowski, A., Pietrucha-Dutczak, M., Kaarniranta, K., Lewin-Kowalik, J., 2014. A rat
experimental model of glaucoma incorporating rapid-onset elevation of intra-
ocular pressure. Sci. Rep. 4, 5910.
Sobrado-Calvo, P., Vidal-Sanz, M., Villegas-Perez, M.P., 2007. Rat retinal microglial
cells under normal conditions, after optic nerve section, and after optic nerve
section and intravitreal injection of trophic factors or macrophage inhibitory
factor. J. Comp. Neurol. 501, 866e878.
Sofroniew, M.V., 2009. Molecular dissection of reactive astrogliosis and glial scar
formation. Trends Neurosci. 32, 638e647.
Song, W.T., Zhang, X.Y., Xia, X.B., 2013. Atoh7 promotes the differentiation of retinal
stem cells derived from Muller cells into retinal ganglion cells by inhibiting
Notch signaling. Stem. Cell Res. Ther. 4, 94.
Stasi, K., Nagel, D., Yang, X., Wang, R.F., Ren, L., Podos, S.M., Mittag, T., Danias, J.,
2006. Complement component 1Q (C1Q) upregulation in retina of murine,
primate, and human glaucomatous eyes. Invest. Ophthalmol. Vis. Sci. 47,
1024e1029.
Steele, M.R., Inman, D.M., Calkins, D.J., Horner, P.J., Vetter, M.L., 2006. Microarray
analysis of retinal gene expression in the DBA/2J model of glaucoma. Invest.
Ophthalmol. Vis. Sci. 47, 977e985.
Stephan, A.H., Barres, B.A., Stevens, B., 2012. The complement system: an unex-
pected role in synaptic pruning during development and disease. Annu. Rev.
Neurosci. 35, 369e389.
Stevens, B., 2008. Neuron-astrocyte signaling in the development and plasticity of
neural circuits. Neurosignals 16, 278e288.
Stevens, B., Allen, N.J., Vazquez, L.E., Howell, G.R., Christopherson, K.S., Nouri, N.,
Micheva, K.D., Mehalow, A.K., Huberman, A.D., Stafford, B., Sher, A., Litke, A.M.,
Lambris, J.D., Smith, S.J., John, S.W., Barres, B.A., 2007. The classical complement
cascade mediates CNS synapse elimination. Cell 131, 1164e1178.
Stone, J., Dreher, Z., 1987. Relationship between astrocytes, ganglion cells and
vasculature of the retina. J. Comp. Neurol. 255, 35e49.
Stone, J., Itin, A., Alon, T., Pe'er, J., Gnessin, H., Chan-Ling, T., Keshet, E., 1995.
Development of retinal vasculature is mediated by hypoxia-induced vascular
endothelial growth factor (VEGF) expression by neuroglia. J. Neurosci. 15,
4738e4747.
Stone, J., Maslim, J., Valter-Kocsi, K., Mervin, K., Bowers, F., Chu, Y., Barnett, N.,
Provis, J., Lewis, G., Fisher, S.K., Bisti, S., Gargini, C., Cervetto, L., Merin, S., Peer, J.,
1999. Mechanisms of photoreceptor death and survival in mammalian retina.
39
Prog. Retin. Eye Res. 18, 689e735.
Sullivan, L.S., Koboldt, D.C., Bowne, S.J., Lang, S., Blanton, S.H., Cadena, E., Avery, C.E.,
Lewis, R.A., Webb-Jones, K., Wheaton, D.H., Birch, D.G., Coussa, R., Ren, H.,
Lopez, I., Chakarova, C., Koenekoop, R.K., Garcia, C.A., Fulton, R.S., Wilson, R.K.,
Weinstock, G.M., Daiger, S.P., 2014. A dominant mutation in hexokinase 1 (HK1)
causes retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 55, 7147e7158.
Takeda, M., Takamiya, A., Jiao, J.W., Cho, K.S., Trevino, S.G., Matsuda, T., Chen, D.F.,
2008. Alpha-aminoadipate induces progenitor cell properties of Müller glia in
adult mice. Invest. Ophthalmol. Vis. Sci. 49, 1142e1150.
Tang, J., Du, Y., Petrash, J.M., Sheibani, N., Kern, T.S., 2013. Deletion of aldose
reductase from mice inhibits diabetes-induced retinal capillary degeneration
and superoxide generation. PLoS One 8, e62081.
Tezel, G., Yang, X., Luo, C., Cai, J., Powell, D.W., 2012. An astrocyte-specific proteomic
approach to inflammatory responses in experimental rat glaucoma. Invest.
Ophthalmol. Vis. Sci. 53, 4220e4233.
Thanos, S., Kacza, J., Seeger, J., Mey, J., 1994. Old dyes for new scopes: the
phagocytosis-dependent long-term fluorescence labelling of microglial cells
in vivo. Trends Neurosci. 17, 177e182.
Toft-Kehler, A.K., Skytt, D.M., Poulsen, K.A., Braendstrup, C.T., Gegelashvili, G.,
Waagepetersen, H., Kolko, M., 2014. Limited energy supply in Muller cells alters
glutamate uptake. Neurochem. Res. 39, 941e949.
Tolentino, M.J., McLeod, D.S., Taomoto, M., Otsuji, T., Adamis, A.P., Lutty, G.A., 2002.
Pathologic features of vascular endothelial growth factor-induced retinopathy
in the nonhuman primate. Am. J. Ophthalmol. 133, 373e385.
Tran, T.L., Bek, T., la Cour, M., Nielsen, S., Prause, J.U., Hamann, S., Heegaard, S., 2014.
Altered aquaporin expression in glaucoma eyes. APMIS 122, 772e780.
Tremblay, M.E., Lowery, R.L., Majewska, A.K., 2010. Microglial interactions with
synapses are modulated by visual experience. PLoS Biol. 8, e1000527.
Tserentsoodol, N., Sztein, J., Campos, M., Gordiyenko, N.V., Fariss, R.N., Lee, J.W.,
Fliesler, S.J., Rodriguez, I.R., 2006. Uptake of cholesterol by the retina occurs
primarily via a low density lipoprotein receptor-mediated process. Mol. Vis. 12,
1306e1318.
Tuo, J., Bojanowski, C.M., Zhou, M., Shen, D., Ross, R.J., Rosenberg, K.I., Cameron, D.J.,
Yin, C., Kowalak, J.A., Zhuang, Z., Zhang, K., Chan, C.C., 2007. Murine ccl2/cx3cr1
deficiency results in retinal lesions mimicking human age-related macular
degeneration. Invest. Ophthalmol. Vis. Sci. 48, 3827e3836.
Turner, D.L., Cepko, C.L., 1987. A common progenitor for neurons and glia persists in
rat retina late in development. Nature 328, 131e136.
Uckermann, O., Vargova, L., Ulbricht, E., Klaus, C., Weick, M., Rillich, K.,
Wiedemann, P., Reichenbach, A., Sykova, E., Bringmann, A., 2004. Glutamate-
evoked alterations of glial and neuronal cell morphology in the guinea pig
retina. J. Neurosci. 24, 10149e10158.
Uckermann, O., Wolf, A., Kutzera, F., Kalisch, F., Beck-Sickinger, A.G., Wiedemann, P.,
Reichenbach, A., Bringmann, A., 2006. Glutamate release by neurons evokes a
purinergic inhibitory mechanism of osmotic glial cell swelling in the rat retina:
activation by neuropeptide Y. J. Neurosci. Res. 83, 538e550.
Ueki, Y., Karl, M.O., Sudar, S., Pollak, J., Taylor, R.J., Loeffler, K., Wilken, M.S.,
Reardon, S., Reh, T.A., 2012. P53 is required for the developmental restriction in
Muller glial proliferation in mouse retina. Glia 60, 1579e1589.
Ueki, Y., Reh, T.A., 2013. EGF stimulates Muller glial proliferation via a BMP-
dependent mechanism. Glia 61, 778e789.
Uga, S., Smelser, 1973. Comparative study of the fine structure of retinal Muller cells
in various vertebrates. Invest. Ophthalmol. 12, 434e448.
Ullian, E.M., Sapperstein, S.K., Christopherson, K.S., Barres, B.A., 2001. Control of
synapse number by glia. Science 291, 657e661.
Unterlauft, J.D., Claudepierre, T., Schmidt, M., Müller, K., Yafai, Y., Wiedemann, P.,
Reichenbach, A., Eichler, W., 2014. Enhanced survival of retinal ganglion cells is
mediated by Müller glial cell-derived PEDF. Exp. Eye Res. 127, 206e214.
Unterlauft, J.D., Eichler, W., Kuhne, K., Yang, X.M., Yafai, Y., Wiedemann, P.,
Reichenbach, A., Claudepierre, T., 2012. Pigment epithelium-derived factor
released by Muller glial cells exerts neuroprotective effects on retinal ganglion
cells. Neurochem. Res. 37, 1524e1533.
Valamanesh, F., Monnin, J., Morand-Villeneuve, N., Michel, G., Zaher, M., Miloudi, S.,
Chemouni, D., Jeanny, J.C., Versaux-Botteri, C., 2013. Nestin expression in the
retina of rats with inherited retinal degeneration. Exp. Eye Res. 110, 26e34.
Vecino, E., 2008. Animal models in the study of the glaucoma: past, present and
future. Arch. Soc. Espanola Oftalmol. 83, 517e519.
Vecino, E., Acera, A., 2015. Development and programed cell death in the eye. Int. J.
Dev. Biol. http://dx.doi.org/ijdb.150070ev (in press).
Vecino, E., Caminos, E., Becker, E., Martin-Zanca, D., Osborne, N., 1998a. Expression
of neurotrophins and their receptors within the glial cells of retina and optic
nerve. In: Castellano, B., Gonz
alez, B., Nieto-Sampedro, M. (Eds.), Understanding
Glial Cells. Kluwer Academic Publisher, pp. 149e166.
Vecino, E., Caminos, E., Ugarte, M., Martin-Zanca, D., Osborne, N.N., 1998b. Immu-
nohistochemical distribution of neurotrophins and their receptors in the rat
retina and the effects of ischemia and reperfusion. Gen. Pharmacol. 30,
305e314.
Vecino, E., Galdos, M., Bayon, A., Rodriguez, F.D., Mico, C., Sharma, S.C., 2015a.
Elevated intraoucular pressure induces ultrastructural changes in the trabecular
meshwork. J. Citol. Histol. http://dx.doi.org/10.4172/2157-7099.S3-007 (in press).
Vecino, E., Garcia-Crespo, D., Garcia, M., Martinez-Millan, L., Sharma, S.C.,
Carrascal, E., 2002. Rat retinal ganglion cells co-express brain derived neuro-
trophic factor (BDNF) and its receptor TrkB. Vis. Res. 42, 151e157.
Vecino, E., Heller, J.P., Veiga-Crespo, P., Martin, K.R., Fawcett, J., 2015b. Influence of
different extracellular matrix components on the expression of integrins and
40 E. Vecino et al. / Progress in Retinal and Eye Research 51 (2016) 1e40
regeneration of adult retinal ganglion cells. PLoS One (in press).
Vecino, E., Sharma, S., 2011. Glaucoma animal models. In: Rumelt, S. (Ed.), Glaucoma
e Basic and Clinical Concepts. In Tech, pp. 319e334.
Vecino, E., Ugarte, M., Nash, M.S., Osborne, N.N., 1999. NMDA induces BDNF
expression in the albino rat retina in vivo. Neuroreport 10, 1103e1106.
Vessey, K.A., Greferath, U., Aplin, F.P., Jobling, A.I., Phipps, J.A., Ho, T., De Iongh, R.U.,
Fletcher, E.L., 2014. Adenosine triphosphate-induced photoreceptor death and
retinal remodeling in rats. J. Comp. Neurol. 522, 2928e2950.
Villacampa, N., Almolda, B., Gonzalez, B., Castellano, B., 2013. Tomato lectin histo-
chemistry for microglial visualization. Methods Mol. Biol. 1041, 261e279.
Vogler, S., Grosche, A., Pannicke, T., Ulbricht, E., Wiedemann, P., Reichenbach, A.,
Bringmann, A., 2013. Hypoosmotic and glutamate-induced swelling of bipolar
cells in the rat retina: comparison with swelling of Müller glial cells.
J. Neurochem. 126, 372e381.
Voskuhl, R.R., Peterson, R.S., Song, B., Ao, Y., Morales, L.B., Tiwari-Woodruff, S.,
Sofroniew, M.V., 2009. Reactive astrocytes form scar-like perivascular barriers
to leukocytes during adaptive immune inflammation of the CNS. J. Neurosci. 29,
11511e11522.
Wahl, V., Vogler, S., Grosche, A., Pannicke, T., Ueffing, M., Wiedemann, P.,
Reichenbach, A., Hauck, S.M., Bringmann, A., 2013. Osteopontin inhibits osmotic
swelling of retinal glial (Muller) cells by inducing release of VEGF. Neuroscience
246, 59e72.
Wang, A.G., Yen, M.Y., Hsu, W.M., Fann, M.J., 2006. Induction of vitronectin and
integrin alphav in the retina after optic nerve injury. Mol. Vis. 12, 76e84.
Wang, B., Xiao, Z., Chen, B., Han, J., Gao, Y., Zhang, J., Zhao, W., Wang, X., Dai, J., 2008.
Nogo-66 promotes the differentiation of neural progenitors into astroglial
lineage cells through mTOR-STAT3 pathway. PLoS One 3, e1856.
Wang, K., Peng, B., Lin, B., 2014. Fractalkine receptor regulates microglial neuro-
toxicity in an experimental mouse glaucoma model. Glia 62, 1943e1954.
Wang, L., Chen, K., Liu, K., Zhou, Y., Zhang, T., Wang, B., Mi, M., 2015. DHA inhibited
AGEs-induced retinal microglia activation via suppression of the PPARgamma/
NFkappaB pathway and reduction of signal transducers in the AGEs/RAGE Axis
recruitment into lipid rafts. Neurochem. Res.
Wang, L.L., Chen, H., Huang, K., Zheng, L., 2012. Elevated histone acetylations in
Muller cells contribute to inflammation: a novel inhibitory effect of minocy-
cline. Glia 60, 1896e1905.
Wang, M., Wong, W.T., 2014. Microglia-Muller cell interactions in the retina. Adv.
Exp. Med. Biol. 801, 333e338.
Watanabe, T., Raff, M.C., 1988. Retinal astrocytes are immigrants from the optic
nerve. Nature 332, 834e837.
Wieghofer, P., Knobeloch, K.P., Prinz, M., 2014. Genetic Targeting of Microglia. Wiley
Periodicals, Inc.
Winkler, B.S., Arnold, M.J., Brassell, M.A., Puro, D.G., 2000. Energy metabolism in
human retinal Muller cells. Invest. Ophthalmol. Vis. Sci. 41, 3183e3190.
Wohl, S.G., Schmeer, C.W., Isenmann, S., 2012. Neurogenic potential of stem/
progenitor-like cells in the adult mammalian eye. Prog. Retin. Eye Res. 31,
213e242.
Wolburg, H., 1981. Myelination and remyelination in the regenerating visual system
of the goldfish. Exp. Brain Res. 43, 199e206.
Wolter, J.R., 1956. Special astrocytes in the inner surface of the retina of the human
eye. Klin. Monatsblatter Augenheilkd. Augenarztl. Fortbild. 129, 224e230.
Wong, V.H., Vingrys, A.J., Bui, B.V., 2011. Glial and neuronal dysfunction in
streptozotocin-induced diabetic rats. J. Ocul. Biol. Dis. Infor. 4, 42e50.
Wurm, A., Iandiev, I., Hollborn, M., Wiedemann, P., Reichenbach, A.,
Zimmermann, H., Bringmann, A., Pannicke, T., 2008a. Purinergic receptor acti-
vation inhibits osmotic glial cell swelling in the diabetic rat retina. Exp. Eye Res.
87, 385e393.
Wurm, A., Pannicke, T., Iandiev, I., Francke, M., Hollborn, M., Wiedemann, P.,
Reichenbach, A., Osborne, N.N., Bringmann, A., 2011. Purinergic signaling
involved in Müller cell function in the mammalian retina. Prog. Retin. Eye Res.
30, 324e342.
Wurm, A., Pannicke, T., Wiedemann, P., Reichenbach, A., Bringmann, A., 2008b. Glial
cell derived glutamate mediates autocrine cell volume regulation in the retina:
activation by VEGF. J. Neurochem. 104, 386e399.
Xie, B., Jiao, Q., Cheng, Y., Zhong, Y., Shen, X., 2012. Effect of pigment epithelium-
derived factor on glutamate uptake in retinal Muller cells under high-glucose
conditions. Invest. Ophthalmol. Vis. Sci. 53, 1023e1032.
Xu, F., Wei, Y., Lu, Q., Zheng, D., Zhang, F., Gao, E., Wang, N., 2009. Immunohisto-
chemical localization of sortilin and p75(NTR) in normal and ischemic rat
retina. Neurosci. Lett. 454, 81e85.
Xu, H., Dawson, R., Forrester, J.V., Liversidge, J., 2007. Identification of novel den-
dritic cell populations in normal mouse retina. Invest. Ophthalmol. Vis. Sci. 48,
1701e1710.
Xue, L.P., Lu, J., Cao, Q., Hu, S., Ding, P., Ling, E.A., 2006a. Muller glial cells express
nestin coupled with glial fibrillary acidic protein in experimentally induced
glaucoma in the rat retina. Neuroscience 139, 723e732.
Xue, L.P., Lu, J., Cao, Q., Kaur, C., Ling, E.A., 2006b. Nestin expression in Muller glial
cells in postnatal rat retina and its upregulation following optic nerve tran-
section. Neuroscience 143, 117e127.
Xue, W., Cojocaru, R.I., Dudley, V.J., Brooks, M., Swaroop, A., Sarthy, V.P., 2011. Ciliary
neurotrophic factor induces genes associated with inflammation and gliosis in
the retina: a gene profiling study of flow-sorted, Muller cells. PLoS One 6,
e20326.
Yafai, Y., Iandiev, I., Lange, J., Unterlauft, J.D., Wiedemann, P., Bringmann, A.,
Reichenbach, A., Eichler, W., 2014. Müller glial cells inhibit proliferation of
retinal endothelial cells via TGF-b2 and smad signaling. Glia 62, 1476e1485.
Yafai, Y., Iandiev, I., Lange, J., Yang, X.M., Wiedemann, P., Bringmann, A., Eichler, W.,
2013. Basic fibroblast growth factor contributes to a shift in the Angioregulatory
activity of retinal glial (Müller cells). PLoS One 8, e68773.
Yafai, Y., Lange, J., Wiedemann, P., Reichenbach, A., Eichler, W., 2007. Pigment
epithelium-derived factor acts as an opponent of growth-stimulatory factors in
retinal glial-endothelial cell interactions. Glia 55, 642e651.
Yan, Q., Li, Y., Hendrickson, A., Sage, E.H., 2001. Regulation of retinal capillary cells
by basic fibroblast growth factor, vascular endothelial growth factor, and hyp-
oxia. In Vitro Cell Dev. Biol. Anim. 37, 45e49.
Yang, L., Xu, Y., Li, W., Yang, B., Yu, S., Zhou, H., Yang, C., Xu, F., Wang, J., Gao, Y.,
Huang, Y., Lu, L., Liang, X., 2014. Diacylglycerol Kinase (DGK) Inhibitor II
(R59949) could suppress retinal neovascularization and protect retinal astro-
cytes in an oxygen-induced retinopathy model. J. Mol. Neurosci.
Yao, J., Sun, X., Wang, Y., Xu, G., Qian, J., 2007. Math5 promotes retinal ganglion cell
expression patterns in retinal progenitor cells. Mol. Vis. 13, 1066e1072.
Ye, X., Xu, G., Chang, Q., Fan, J., Sun, Z., Qin, Y., Jiang, A.C., 2010. ERK1/2 signaling
pathways involved in VEGF release in diabetic rat retina. Invest. Ophthalmol.
Vis. Sci. 51, 5226e5233.
Yoshida, S., Sotozono, C., Ikeda, T., Kinoshita, S., 2001. Interleukin-6 (IL-6) produc-
tion by cytokine-stimulated human Muller cells. Curr. Eye Res. 22, 341e347.
Yu, A.L., Welge-Lussen, U., 2013. Antioxidants reduce TGF-beta2-induced gene ex-
pressions in human optic nerve head astrocytes. Acta Ophthalmol. 91, e92e98.
Yu, D.Y., Cringle, S.J., Balaratnasingam, C., Morgan, W.H., Yu, P.K., Su, E.N., 2013.
Retinal ganglion cells: energetics, compartmentation, axonal transport, cyto-
skeletons and vulnerability. Prog. Retin. Eye Res. 36, 217e246.
Yu, J., Chen, C., Wang, J., Cheng, Y., Wu, Q., Zhong, Y., Shen, X., 2012. In vitro effect of
adenosine on the mRNA expression of Kir 2.1 and Kir 4.1 channels in rat retinal
Muller cells at elevated hydrostatic pressure. Exp. Ther. Med. 3, 617e620.
Zack, D.J., 2000. Neurotrophic rescue of photoreceptors: are Müller cells the me-
diators of survival? Neuron 26, 285e286.
Zamanian, J.L., Xu, L., Foo, L.C., Nouri, N., Zhou, L., Giffard, R.G., Barres, B.A., 2012.
Genomic analysis of reactive astrogliosis. J. Neurosci. 32, 6391e6410.
Zeng, H., Ding, M., Chen, X.X., Lu, Q., 2014. Microglial NADPH oxidase activation
mediates rod cell death in the retinal degeneration in rd mice. Neuroscience
275, 54e61.
Zeng, H.Y., Zhu, X.A., Zhang, C., Yang, L.P., Wu, L.M., Tso, M.O., 2005. Identification of
sequential events and factors associated with microglial activation, migration,
and cytotoxicity in retinal degeneration in rd mice. Invest. Ophthalmol. Vis. Sci.
46, 2992e2999.
Zhang, S., Li, W., Wang, W., Zhang, S.S., Huang, P., Zhang, C., 2013. Expression and
activation of STAT3 in the astrocytes of optic nerve in a rat model of transient
intraocular hypertension. PLoS One 8, e55683.
Zhang, X., Cheng, M., Chintala, S.K., 2004. Kainic acid-mediated upregulation of
matrix metalloproteinase-9 promotes retinal degeneration. Invest. Ophthalmol.
Vis. Sci. 45, 2374e2383.
Zhao, J.J., Ouyang, H., Luo, J., Patel, S., Xue, Y., Quach, J., Sfeir, N., Zhang, M., Fu, X.,
Ding, S., Chen, S., Zhang, K., 2014a. Induction of retinal progenitors and neurons
from mammalian Müller glia under defined conditions. J. Biol. Chem. 289,
11945e11951.
Zhao, Z., Xu, P., Jie, Z., Zuo, Y., Yu, B., Soong, L., Sun, J., Chen, Y., Cai, J., 2014b.
Gammadelta T cells as a major source of IL-17 production during age-
dependent RPE degeneration. Invest. Ophthalmol. Vis. Sci. 55, 6580e6589.
Zheng, X.R., Zhang, S.S., Yin, F., Tang, J.L., Yang, Y.J., Wang, X., Zhong, L., 2012.
Neuroprotection of VEGF-expression neural stem cells in neonatal cerebral
palsy rats. Behav. Brain Res. 230, 108e115.
Zhou, X., Li, F., Kong, L., Chodosh, J., Cao, W., 2009. Anti-inflammatory effect of
pigment epithelium-derived factor in DBA/2J mice. Mol. Vis. 15, 438e450.
Zieger, M., Ahnelt, P.K., Uhrin, P., 2014. CX3CL1 (fractalkine) protein expression
in normal and degenerating mouse retina: in vivo studies. PLoS One 9,
e106562.