Progress in brain research, volume 220

Preface: New Trends in Basicand Clinical Research of Glaucoma: A Neurodegenerative Disease of the Visual System Part A Glaucoma, the second leading cause of blindness in the world, is ch

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Mark Bear, Cambridge, USA.

Medicine & Translational NeuroscienceHamed Ekhtiari, Tehran, Iran

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Jessica Agostinone

Department of Neuroscience, and Centre de Recherche du Centre Hospitalier

de l’Universite´ de Montre´al (CRCHUM), University of Montreal, Montreal, QC,

Canada

Marta Agudo-Barriuso

Departamento de Oftalmologı´a, Universidad de Murcia and Instituto Murciano de

Investigacio´n Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain

Luis Alarco´n-Martı´nez

Marcelino Avile´s-Trigueros

Giacinto Bagetta

Department of Pharmacy and Health and Nutritional Sciences, Section of

Preclinical and Translational Pharmacology, University of Calabria, Arcavacata

di Rende, Italy; University Consortium for Adaptive Disorders and Head Pain

(UCHAD), Section of Neuropharmacology of Normal and Pathological Neuronal

Plasticity, University of Calabria, Arcavacata di Rende, Italy

Claudio Bucolo

Department of Biomedical and Biotechnological Sciences, Section of

Pharmacology, University of Catania, Catania, Italy

Karolien Castermans

Amakem Therapeutics, Diepenbeek, Belgium

Shenton S.L Chew

NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation

Trust and UCL Institute of Ophthalmology, London, UK

Maria Tiziana Corasaniti

Department of Health Sciences, University “Magna Graecia” of Catanzaro,

Catanzaro, Italy

Rosa de Hoz

Instituto de Investigaciones Oftalmolo´gicas Ramo´n Castroviejo, Facultad de

O´ptica y Optometrı´a, Universidad Complutense de Madrid, Spain

Adriana Di Polo

Department of Neuroscience, and Centre de Recherche du Centre Hospitalier

de l’Universite´ de Montre´al (CRCHUM), University of Montreal, Montreal, QC,

Canada

v

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Filippo Drago

Diego Garcı´a-Ayuso

Departamento de Oftalmologıá, Universidad de Murcia and Instituto Murciano deInvestigacioń Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, SpainJose´ J Garcıá-Medina

Ophthalmic Research Unit “Santiago Grisolı´a”, University Hospital Dr Peset,Valencia; Department of Ophthalmology, University Hospital Reina Sofia, andDepartment of Ophthalmology and Optometry, University of Murcia, Murcia,Spain

Neeru Gupta

Department of Ophthalmology and Vision Sciences; Department of LaboratoryMedicine and Pathobiology, University of Toronto; Keenan Research Centre forBiomedical Science, and Glaucoma and Nerve Protection Unit, St Michael’sHospital, Toronto, ON, Canada

Manuel Jime´nez-Lo´pez

Departamento de Oftalmologı´a, Universidad de Murcia and Instituto Murciano deInvestigacio´n Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, SpainNele Kindt

Amakem Therapeutics, Diepenbeek, Belgium

vi Contributors

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Alessandra Martins

Discipline of Clinical Ophthalmology and Eye Health, University of Sydney, and

Sydney Eye Hospital, Sydney, NSW, Australia

Lieve Moons

Research Group of Neural Circuit Development and Regeneration, KU Leuven,

Leuven, Belgium

Luigi Antonio Morrone

Department of Pharmacy and Health and Nutritional Sciences, Section of

Preclinical and Translational Pharmacology, University of Calabria, Arcavacata di

Rende, Italy; University Consortium for Adaptive Disorders and Head Pain

(UCHAD), Section of Neuropharmacology of Normal and Pathological Neuronal

Plasticity, University of Calabria, Arcavacata di Rende, Italy

Francisco M Nadal-Nicola´s

Carlo Nucci

Ophthalmology Unit, Department of Experimental Medicine and Surgery,

University of Rome Tor Vergata, Rome, Italy

Arturo Ortı´n-Martı´nez

Craig Pearson

John van Geest Centre for Brain Repair, University of Cambridge; Cambridge

NIHR Biomedical Research Centre, Cambridge, UK, and National Heart, Lung

and Blood Institute, National Institutes of Health, Bethesda, MD, USA

Maria D Pinazo-Dura´n

Ophthalmic Research Unit “Santiago Grisolı´a”, University Hospital Dr Peset, and

Department of Surgery/Ophthalmology, Faculty of Medicine and Odontology,

University of Valencia, Valencia, Spain

Chiara Bianca Maria Platania

Harry A Quigley

Glaucoma Center of Excellence, Wilmer Institute, Johns Hopkins University

School of Medicine, Baltimore, MD, USA

Ana I Ramı´rez

Instituto de Investigaciones Oftalmolo´gicas Ramo´n Castroviejo, Facultad de

O´ptica y Optometrı´a, Universidad Complutense de Madrid, Spain

Jose´ M Ramirez

Instituto de Investigaciones Oftalmolo´gicas Ramo´n Castroviejo, Departamento de

Oftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,

Spain

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Blanca Rojas

Instituto de Investigaciones Oftalmolo´gicas Ramo´n Castroviejo, Departamento deOftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,Spain

Giovanni Luca Romano

Instituto de Investigaciones Oftalmolo´gicas Ramo´n Castroviejo, UniversidadComplutense de Madrid, Spain

Salvatore Salomone

Alberto Trivin˜o

Instituto de Investigaciones Oftalmolo´gicas Ramo´n Castroviejo, Departamento deOftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,Spain

viii Contributors

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Francisco J Valiente-Soriano

Laboratorio de Oftalmologı´a Experimental, Departamento de Oftalmologı´a,

Facultad de Medicina, Universidad de Murcia, and Departamento de

Oftalmologı´a, Universidad de Murcia and Instituto Murciano de Investigacio´n

Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain

Tine Van Bergen

Laboratory of Ophthalmology, KU Leuven, Leuven, Belgium

Sarah Van de Velde

Laboratory of Ophthalmology, KU Leuven, Leuven, Belgium

Evelien Vandewalle

Laboratory of Ophthalmology, KU Leuven, and Department of Ophthalmology,

University Hospitals Leuven (UZ Leuven), Leuven, Belgium

Manuel Vidal-Sanz

Maria P Villegas-Pe´rez

Yeni Yucel

Department of Ophthalmology and Vision Sciences; Department of Laboratory

Medicine and Pathobiology, University of Toronto; Keenan Research Centre

for Biomedical Science; Ophthalmic Pathology Laboratory, University of Toronto,

St Michael’s Hospital, and Faculty of Engineering & Architectural Science,

Ryerson University, Toronto, ON, Canada

Vicente Zano´n-Moreno

Ophthalmic Research Unit “Santiago Grisolı´a”, University Hospital Dr Peset, and

Department of Surgery/Ophthalmology, Faculty of Medicine and Odontology,

University of Valencia, Valencia, Spain

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Preface: New Trends in Basic

and Clinical Research of Glaucoma:

A Neurodegenerative Disease of the

Visual System Part A

Glaucoma, the second leading cause of blindness in the world, is characterized by

progressive retinal ganglion cell (RGC) axons degeneration and death leading to

typ-ical optic nerve head damage and distinctive visual field defects This disease is a

chronic optic neuropathy most often associated with increased intraocular pressure

and age as main risk factors Defective axonal transport, trophic factor withdrawal,

and neuroinflammation are emerging as important pathophysiological factors

Despite the limited value of the animal models in recapitulating the

pathophys-iology of the disease, these have allowed determinants involved in RGC apoptosis to

be dissected Under conditions of glutamate homeostasis disruption, excitotoxicity

ensues and this causes neuronal damage implicating oxidative stress Free radical

species accumulation can cause RGC death by inhibition of key enzymes of the

tri-carboxylic acid cycle, the mitochondrial electron transport chain, and mitochondrial

calcium homeostasis, leading to defective energy metabolism

Accordingly, in glaucomatous patients a significant decrease in the total

antiox-idant capacity has been reported along with increased end-products of lipid

perox-idation, among other putative markers Several interventions find their rational in the

causative role of oxidative stress in RGC death, though these have limited or no

clin-ical proof

Experimental data indicate that axonal injury triggers rapid structural alterations

in RGC dendritic arbors, prior to manifest axonal loss, leading to synaptic

rearrange-ments and functional deficits

Tissue remodeling occurring in glaucoma may cause biomechanical and

micro-structural changes that are likely to alter the mechanical environment of the optic

nerve head and may contribute to axonal damage Indeed, experimental evidence

fol-lowing laser photocoagulation demonstrates that the volume occupied by retinotectal

afferents is halved, ocular hypertension affects selectively projecting neurons (e.g.,

RGC), and intraocular administration of BDNF results in increased RGC survival

These data are at variance with changes in other cells/sectors of the retina for the

proportion of the cell loss, for its diffuse and not sectorial topography, for it does

not respond to BDNF neuroprotection, and for progressive functional and

morpho-logical alterations there occur

Most of the data in the literature have been gathered employing experimental

models of unilateral glaucoma and using the normotensive contralateral eye as the

normal control Interestingly, some studies have recently reported the activation

xix

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of the retinal macroglia and microglia in the uninjured eye along with important servations implicating innate and adaptive immunity The latter data support a rolefor blood–retina barrier disruption in the pathophysiology of glaucoma-associatedneurodegenerative process other than simply suggesting that the eye contralateral

ob-to experimental glaucoma cannot be a true control

Experimental data do support the hypothesis that autophagy might participate inthe process leading to RGC death though the precise role awaits to be clarified Infact, evidence shows that downregulation of autophagy-related genes (Atg5, Atg7,and BECN1) in normal human aging brain has been reported On the contrary, a re-cent study analyzing LC3 and p62 levels in fresh TM from human donors reportedlower levels of p62 and increased LC3II/LC3I ratio in subjects older than 60 yearssuggesting an age-related upregulation of autophagy in the TM A marked reduction

in macroautophagy activity in the aged retina that is associated,in vitro and in vivo,with a sustained upregulation of the chaperone-mediated autophagy in the compro-mised cells has been recently noticed Accordingly, age-related dysfunction ofautophagy in the retina might represent another determinant for glaucomaprogression

Indeed, association of glaucoma with age-related neurodegenerative diseasesstems from these sharing similar miRNAs regulated transduction pathways (see alsoPart B for additional evidence) In fact, by means ofin silico approaches and access

to bioinformatic resources, deregulated miRNAs in glaucoma, in age-related lar degeneration (AMD) and Alzheimer’s disease (AD), respectively, have beenfound Actually, 88 predicted miRNAs are common to glaucoma and AMD;

macu-19 are common to glaucoma and AD; and 9 are common to AMD and AD Thesefindings provide a valuable hint to assess deregulation of specific miRNA as poten-tial biomarkers and therapeutic targets, in glaucoma and other neurodegenerativediseases by means of preclinical and clinical studies

The wealth of the above-mentioned data in conjunction with important newsemerging from clinical genetics and cell therapy technology is deeply discussed

by authoritative, world-widely recognized, scientists in this issue (Part A) ofProgress in Brain Research dedicated to glaucoma To them is addressed our sincereacknowledgment for making the issue a success Also, our thanks go to the skillfultechnical collaboration of individuals belonging to the Production Department ofElsevier We are especially indebted to Shellie Bryant for her continuous and highlyqualified editorial assistance from the very beginning of this venture

The EditorsGiacinto Bagetta and Carlo Nucci

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Retinal neurodegeneration in

Manuel Vidal-Sanz1,2, Francisco J Valiente-Soriano1, Arturo Ortı´n-Martı´nez1,

Francisco M Nadal-Nicola´s1, Manuel Jime´nez-Lo´pez, Manuel Salinas-Navarro,

Luis Alarcoń-Martıńez, Diego Garcıá-Ayuso, Marcelino Avile´s-Trigueros,

Marta Agudo-Barriuso, Maria P Villegas-Pe´rez

Departamento de Oftalmologı´a, Universidad de Murcia and Instituto Murciano de Investigacio´n

Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain

2 Corresponding author: Tel.: +34-868-884330; Fax: +34-868-883962,

e-mail address: manuel.vidal@um.es

Abstract

In rats and mice, limbar tissues of the left eye were laser-photocoagulated (LP) and ocular

hypertension (OHT) effects were investigated 1 week to 6 months later

To investigate the innermost layers, retinas were examined in wholemounts using tracing

from the superior colliculi to identify retinal ganglion cells (RGCs) with intact retrograde

ax-onal transport, melanopsin immunodetection to identify intrinsically photosensitive RGCs

(m+RGC), Brn3a immunodetection to identify most RGCs but not m+RGCs, RECA1

immu-nodetection to examine the inner retinal vessels, and DAPI staining to detect all nuclei in the

GC layer The outer retinal layers (ORLs) were examined in cross sections analyzed

morpho-metrically or in wholemounts to study S- and L-cones Innervation of the superior colliculi was

examined 10 days to 14 weeks after LP with orthogradely transported cholera toxin subunit B

By 2 weeks, OHT resulted in pie-shaped sectors devoid of FG+RGCs or Brn3a+RGCs but

with large numbers of DAPI+nuclei Brn3a+RGCs were significantly greater than FG+RGCs,

indicating the survival of large numbers of RGCs with their axonal transport impaired The

inner retinal vasculature showed no abnormalities that could account for the sectorial loss

of RGCs m+RGCs decreased to approximately 50–51% in a diffuse loss across the retina

Cross sections showed focal areas of degeneration in the ORLs RGC loss at 1 m diminished

to 20–25% and did not progress further with time, whereas the S- and L-cone populations

di-minished progressively up to 6 m The retinotectal projection was reduced by 10 days and did

not progress further LP-induced OHT results in retrograde degeneration of RGCs and

m+RGCs, severe damage to the ORL, and loss of retinotectal terminals

1 Equally contributed to this work.

Progress in Brain Research, Volume 220, ISSN 0079-6123, http://dx.doi.org/10.1016/bs.pbr.2015.04.008

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Laser-induced ocular hypertension, Intrinsically photosensitive melanopsin RGCs, BDNFneuroprotection, Adult rodents, Experimental glaucoma, Axonal transport, Brn3a, Fluorogold,S- and L-cones, Neuronal degeneration

The progressive loss of retinal ganglion cells (RGCs) and their axons with itant insidious defects in the visual field has been the classic hallmark of the glau-comatous optic neuropathies (GONs) (Chauhan et al., 2014; Quigley, 2011;

estab-lished that GONs involve not only the RGC population, the nerve fiber layer of theretina (O’Leary et al., 2012), the optic disc, and optic nerve (ON) head (Chauhan

the primary visual pathway, such as the lateral geniculate nucleus and primaryand secondary visual areas of the cortex (Dekeyster et al., 2015; Garaci et al.,2009; Gupta and Yu¨cel, 2007; Nucci et al., 2013a; Yu¨cel and Gupta, 2008; Yu¨cel

be-come affected (Frezzotti et al., 2014) Here, we review some recent experiments

in adult pigmented mice that have investigated the effects of ocular hypertension(OHT) in the main retinorecipient target nuclei in the brain, the superior colliculus(SC) (Valiente-Soriano et al., 2015a)

One of the main risk factors for glaucoma is elevated intraocular pressure (IOP)above normal levels, and the only one for which there is currently medical treatment;thus, a number of studies have investigated the effects of OHT on the retina and vi-sual system Taking advantage of the anatomy of the aqueous humor draining system

in rodents, several models have been developed to induce OHT (Morrison et al.,

1995), including the episcleral vein cauterization (Garcia-Valenzuela et al., 1995),the injection into episcleral veins of hypertonic saline (Morrison et al., 1997), theadministration into the anterior chamber of microbeads or viscoelastics (Abbott

et al., 2014; El-Danaf and Huberman, 2015; Sappington et al., 2010; Urcola et al.,

2006), and the photocauterization with laser of the perilimbar and episcleral veins

several spontaneous models of experimental glaucoma in mice with a targeted type

I collagen mutation (Aihara et al., 2003) or the DBA/2J mice which develops a mentary glaucoma (Buckingham et al., 2008; Danias et al., 2003; Filippopoulos

pig-et al., 2006; Panagis pig-et al., 2010; Pe´rez de Lara pig-et al., 2014; Reichstein pig-et al.,

2007) In our laboratory, laser photocoagulation (LP) of the limbar tissues hasbeen the method of choice to induce OHT in adult albino rats (Ortı´n-Martı´nez

et al., 2015; Ramı´rez et al., 2010; Salinas-Navarro et al., 2010; Schnebelen et al.,

et al., 2013; Dekeyster et al., 2015; Gallego et al., 2012; Rojas et al., 2014;

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Salinas-Navarro et al., 2009c) or pigmented (Nguyen et al., 2011; Valiente-Soriano

of RGCs, an initial damage to RGC axons somewhere near the ON head, and an

al-teration of the retrograde axoplasmic transport that precedes RGC death (Chidlow

et al., 2011; Martin et al., 2006; Soto et al., 2011; Vidal-Sanz et al., 2012) all of which

are also found in a classic model of glaucoma, the DBA/2J mouse (Buckingham

et al., 2008; Crish et al., 2010; Filippopoulos et al., 2006; Jakobs et al., 2005), thus

making this model relevant to advance our knowledge on the retinal pathology

induced by OHT

In certain glaucoma patients despite the efforts to maintain IOP below certain

levels, RGC loss keeps progressing to blindness This has prompted investigators

to look for alternatives to prevent or slow cell death using neuroprotective drugs

(Almasieh et al., 2012; Nucci et al., 2013b; Russo et al., 2013) Partial and transient

rescue of RGCs against a variety of retinal injuries has been shown with several

neu-roprotective agents (Jehle et al., 2008; Vidal-Sanz et al., 2000, 2001, 2007),

includ-ing brain-derived neurotrophic factor (BDNF), which has been shown to be one

of the most potent RGC neuroprotectants (Di Polo et al., 1998; Galindo-Romero

et al., 2013b; Mansour-Robaey et al., 1994; Peinado-Ramo´n et al., 1996;

to prevent OHT-induced RGC loss (Almasieh et al., 2012; Di Polo et al., 1998; Fu

et al., 2009; Ko et al., 2001; Lebrun-Julien et al., 2008; Martin et al., 2003; Quigley

the neuroprotective effects of BDNF on the population of injured RGCs, including

melanopsin-expressing (m+RGCs) and nonmelanopsin-expressing RGCs (

Valiente-Soriano et al., 2015b)

A number of reports have also indicated that other neurons in the retina, besides

RGCs, are also affected in human and experimental glaucoma Several groups have

documented important molecular, functional, and structural changes in the outer

(outer nuclear and outer segment) retinal layers of the retina in clinical human

glau-coma studies (Choi et al., 2011; Drasdo et al., 2001; Holopigian et al., 1990; Kanis

et al., 2010; Lei et al., 2008, 2011; Nork, 2000; Nork et al., 2000; Panda and Jonas,

1992; Velten et al., 2001; Werner et al., 2011), as well as in nonhuman primate (Nork

et al., 2014; Pelzel et al., 2006) and rodent models of glaucoma or OHT (Bayer et al.,

2001; Cuenca et al., 2010; Ferna´ndez-Sa´nchez et al., 2014; Georgiou et al., 2014;

Holcombe et al., 2008; Kong et al., 2009; Korth et al., 1994; Mittag et al., 2000;

from a diminution in the expression of opsins by photoreceptors to the severe loss

of rod and cone photoreceptors with time Here, we review some recent studies

on the effects of OHT on the outer retinal layers (ORLs) in adult rodents

RGCs are comprised of several types; each one devoted to a specific function

and with a clear major target nuclei in the brain Up to now, with few exceptions

retinal degeneration have been studied as a whole Intrinsically photosensitive

RGCs (ipRGCs) mediate a number of nonimage-forming visual functions such as

3

1 Introduction

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photoentrainment of the circadian rhythms, photic suppression of melatonin tion, and pupillary light reflexes (Berson et al., 2002; Hankins et al., 2008; Hattar

melanopsin which can be readily identified with melanopsin antibodies (m+RGCs).This tool provides unique opportunities to examine the responses of one of the manytypes of RGCs against OHT-induced degeneration m+RGCs constitute between 2%and 3% of all RGCs in adult rats (2.5% and 2.7% for albino and pigmented, respec-tively;Galindo-Romero et al., 2013a; Nadal-Nicola´s et al., 2012, 2014) and mice(2.5% and 2.1% for albino and pigmented, respectively; Valiente-Soriano et al.,

number of altered nonvisual-forming functions (Feigl et al., 2011; Kankipati

et al., 2011; Martucci et al., 2014; Nissen et al., 2014; Pe´rez-Rico et al., 2010) over, experimental glaucoma in rats has been shown to present important alterations

More-of the circadian rhythms (de Zavalı´a et al., 2011; Drouyer et al., 2008; Zhang et al.,

2013) Here, we review some recent studies on the effects of OHT on the survival of

m+RGCs and examine the topological distribution as well as their responsiveness tointraocular administration of BDNF

In the following lines, we review some recent studies in our laboratory on rat andmice models of laser-induced OHT (Agudo-Barriuso et al., 2013a; Cuenca et al.,2010; Nadal-Nicola´s et al., 2014; Ortı´n-Martı´nez et al., 2015; Salinas-Navarro

et al., 2009c, 2010; Valiente-Soriano et al., 2015a,b; Vidal-Sanz et al., 2012) Usingmodern techniques to identify and map in the same retinal wholemounts, the generalpopulation of RGCs (nonmelanopsin expressing, identified with Brn3a antibodies),the population of m+RGCs, the population of calretinin-expressing displaced ama-crine cells, the subpopulation of displaced RGCs, the entire cell population in theganglion cell layer (GCL) (identified with DAPI nuclear staining), the nerve fiberlayer of the retina (identified with neurofilament antibodies), and the inner arterialretinal vasculature (identified with RECA1 immunostaining), we have investigatedthe responses of non-m+RGCs to OHT-induced retinal degeneration and neuropro-tection afforded by BDNF and compared them to those of m+RGCs Moreover, wehave examined up to 6 months the effects of OHT on the ORLs in radial cross sec-tions of the retinas as well as in wholemounts, in which we have quantified andmapped the populations of surviving RGCs, S- and L-cones Finally, using choleratoxin B subunit (CTB) as a fine anterograde tracer, we have investigated the fate ofthe retinal terminals in their main target in the brain, the contralateral SC

2.1 ANIMAL HANDLING

Experiments were prepared in accordance with the ARVO, the European Unionguidelines for the use of animals in research, and the Ethical and Animal StudiesCommittee of the University of Murcia (UM) Adult female albino Sprague–Dawley

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(SD) rats (180–230 g) or male Swiss or pigmented C57BL/6 mice (25–35 g) were

obtained from the UM breeding colony and were housed at UM animal facilities

in temperature- and light-controlled rooms (12 h light/dark cycle) with food and

wa-terad libitum Surgeries and IOP measurements were performed under anesthesia

[intraperitoneal (ip) injection of xylazine (10 mg/kg body weight, Rompu´n®; Bayer,

S.A., Barcelona, Spain) and ketamine (60 mg/kg bw, Imalgene; Merial Laboratorios,

Barcelona, Spain)] During recovery, topical ointment (Tobrex®; Alco´n Cusı´, S.A.,

Barcelona, Spain) was applied to prevent corneal desiccation All efforts were taken

to minimize animal suffering and analgesics were administrated during the first

week Animals were sacrificed with an ip overdose of 20% sodium pentobarbital

(Dolethal Vetoquinol®; Especialidades Veterinarias, S.A., Alcobendas, Madrid,

Spain) Recent studies indicate that injury to one eye may produce significant

molecular changes in the intact contralateral eye (Bodeutsch et al., 1999; de Hoz

Rojas et al., 2014); thus for control experiments, naı¨ve (intact) animals were used

2.2 ANIMAL MANIPULATIONS

OHT was achieved by LP (Viridis Ophthalmic Photocoagulator-532 nm laser;

Quan-tel Medical, Clermont-Ferrand, France) of the perilimbal and episcleral vessels

bilaterally prior to, and at 12, 24, 48 h, 3 days, 1 or 2 weeks, 3 or 6 months after LP

with a rebound tonometer (Tono-Lab; Tiolat Oy, Helsinki, Finland) (

all other measurements were obtained at the same time in the morning to avoid

IOP fluctuations due to circadian rhythms (Jia et al., 2000; Krishna et al., 1995;

Moore et al., 1996) or to elevation of the IOP itself (Drouyer et al., 2008) Rat RGCs

were identified with Fluorogold® (FG; Fluorochrome Corp, Denver, CO), while

mice RGCs were identified with the FG analogue hydroxystilbamidine

methanesul-fonate (OHSt; Molecular Probes, Leiden, The Netherlands) which is a small

mole-cule with similar fluorescent and tracer properties to FG (Cheunsuang and Morris,

2005), applied to both superior colliculi (SCi) 1 week before animal processing as

reported in detail (Salinas-Navarro et al., 2009a,b; Vidal-Sanz et al., 2000) To study

the neuroprotective effects of BDNF on the survival of RGCs, 5mg of BDNF

(Peprotech Laboratories, London, UK) or vehicle was intravitreally injected in

the left eye following standard procedures in this laboratory (Vidal-Sanz et al.,

2000) prior to LP of the limbal and episcleral vessels (Valiente-Soriano et al.,

the orthogradely transported tracer CTB was intravitreally injected (1%, diluted in

distilled water; List Biological Laboratories, Campbell, CA) following previously

described protocols that are standard in our laboratory (Avile´s-Trigueros et al.,

2000, 2003; Mayor-Torroglosa et al., 2005; Vidal-Sanz et al., 2002, 2007;

Whiteley et al., 1998)

5

2 Methods

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2.3 TISSUE PROCESSING

Rats or mice were sacrificed and perfused transcardially with 4% paraformaldehyde

in 0.1 M phosphate buffer after a saline rinse

2.3.1 Retinal Wholemounts

Retinas were dissected and prepared as flattened wholemounts maintaining theretinal orientation by making four radial cuts (the deepest in the superior pole) aspreviously described in detail (Nadal-Nicola´s et al., 2009, 2012, 2014, 2015;Salinas-Navarro et al., 2009a,b)

2.3.2 SCi Serial Sections

Brain serial coronal sections (30mm thick) from the level of the anterior thalamus tothe rostral pole of the cerebellum were obtained on a freezing cryostate (Avile´s-Trigueros et al., 2000)

2.3.3 Retinal Cross Sections

Eyes were embedded in paraffin (Garcıá-Ayuso et al., 2010; Ortıń-Martıńez et al.,

2015), and 3-mm-thick cross section cut in the parasagittal plane comprising thesuperior and the inferior retina within the width of the ON head was obtained in amicrotome (Microm HM-340-E; Microm Laborgerate GmbH, Walldorf, Germany)and stained with hematoxylin–eosin (Garcı´a-Ayuso et al., 2010, 2011)

2.4 IMMUNODETECTION AND DAPI STAINING

Immunofluorescence in flat-mounted retinas and cross sections was carried outfollowing previously described methods (Galindo-Romero et al., 2011, 2013a,b;Garcı´a-Ayuso et al., 2010, 2011, 2013; Nadal-Nicola´s et al., 2009, 2012, 2014;

immunodetected by their specific opsin expression (Garcı´a-Ayuso et al., 2013;

immunode-tected with Brn3a (Galindo-Romero et al., 2011; Nadal-Nicola´s et al., 2009,

et al., 2013a; Nadal-Nicola´s et al., 2014; Valiente-Soriano et al., 2014, 2015a,b), placed amacrine cells with calretinin (Ortı´n-Martı´nez et al., 2015), and inner retinalvessels with RECA1 (Valiente-Soriano et al., 2015b) To study all cells in the GCL,retinal wholemounts were stained with DAPI (Vectashield mounting medium withDAPI; Vector Laboratories, Inc., Burlingame, CA) For details about the primaryantibodies employed in this study, seeTable 1

dis-Secondary fluorescent antibodies were donkey anti-goat Alexa Fluor 594, key anti-rabbit Alexa Fluor 488, and donkey anti-mouse Alexa Fluor 488(Molecular Probes, ThermoFisher, Madrid, Spain) All were used at 1:500 dilution.Transported CTB from the retina to the terminals in the SCi was immunolocalizedwith goat anti-CTB antibody and the ABC complex immunoperoxidase method(Vectastain ABC Kit Elite; Vector Laboratories, Burlingame, CA) as previously

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don-described (Avile´s-Trigueros et al., 2000, 2003; Mayor-Torroglosa et al., 2005;

Valiente-Soriano et al., 2015a; Vidal-Sanz et al., 2002, 2007; Whiteley et al., 1998)

2.5 IMAGE ACQUISITION

Micrographs were taken to reconstruct retinal wholemounts or cross sections

follow-ing previously described procedures that are standard in our laboratory (

Galindo-Romero et al., 2011, 2013a; Nadal-Nicola´s et al., 2009; Salinas-Navarro et al.,

(Axioscop 2 Plus; Zeiss Mikroskopie, Jena, Germany) equipped with a

computer-driven motorized stage (ProScan H128 Series; Prior Scientific Instruments,

Cambridge, UK) controlled by image analysis software (Image-Pro Plus, IPP 5.1

for Windows; Media Cybernetics, Silver Spring, MD) Each reconstructed

Table 1 Primary antibodies used in this work

Detection

of Antigen Antibody Source

Working dilution Retinotectal

terminals

Cholera toxin

subunit B

Goat CTB

anti-703 List Biological Laboratories, QuadraTech, Surrey, UK

anti-MCA1321 Serotec, Bionova Scientific, Madrid, Spain

1:200

RGCs Brn3a (Pou4f1) Goat

anti-Brn3a (C-20)

sc-31984 Santa Cruz Biotechnologies, Heidelberg, Germany

1:2500

L-cones Human red/

green opsin

Rabbit opsin red/

anti-green

ab5405 Millipore Iberica, Madrid, Spain

Chemicon-1:1200

S-cones Blue opsin Goat

anti-OPNS1SW (N20)

sc-14363 Santa Cruz Biotechnologies, Heidelberg, Germany

anti-MCA970 Serotec, Bionova Scientific, Madrid, Spain

PA1-780 Pierce, ThermoFisher, Madrid, Spain

AB-N38 Advance Targeting Systems, Thermo Scientific, Madrid, Spain

1:5000

7

2 Methods

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wholemount or cross section was composed of 154 (rat) or 140 (mouse) individualframes captured side by side with no gap or overlap between them with a 10 (rat)

or 20 (mouse) objective (Plan-Neofluar, Zeiss Mikroskopie, Jena, Germany).When required, images were further processed using a graphics editing program(Adobe Photoshop CS 8.0.1; Adobe Systems, Inc., San Jose, CA)

2.6 IMAGE ANALYSIS

FG+RGCs, Brn3a+RGCs, DAPI+nuclei, and L- and S- cones were counted ically, while m+RGCs were counted manually following previously describedmethods (Galindo-Romero et al., 2011, 2013a; Nadal-Nicola´s et al., 2009, 2012,2014; Ortı´n-Martı´nez et al., 2010, 2014; Salinas-Navarro et al., 2009a; Valiente-

using isodensity maps (Galindo-Romero et al., 2011; Nadal-Nicola´s et al., 2009;

nearest m+RGCs was visualized using neighbor maps (Galindo-Romero et al.,2013a; Nadal-Nicola´s et al., 2014; Valiente-Soriano et al., 2014) To study the innerretinal vessels, RECA1 signal was transformed into a black (background) and white(vessels signal) image using the image analysis software IPP (Valiente-Soriano et al.,

photo-montages with a semiautomated routine developed with IPP macrolanguage(Ortı´n-Martı´nez et al., 2015) The area occupied with CTB labeling in the contralat-eral or ipsilateral SC was measured using the image analysis software IPP (Valiente-Soriano et al., 2015a) A polynomial regression line was obtained for each individual

SC and the integral of the function yielded the volume of the SC occupied by intenseCTB labeling in each animal as previously described in detail (Mayor-Torroglosa

et al., 2005; Valiente-Soriano et al., 2015a)

2.7 STATISTICAL ANALYSIS

All data are presented as means with standard deviations Statistical analysis wasdone using SigmaStat® 3.1 for Windows® (SigmaStat® for Windows™ Version3.11; Systat Software, Inc., Richmond, CA) Kruskal–Wallis was used when compar-ing more than two groups and Mann–Whitney when comparing two groups only.Differences were considered significant whenp<0.05

Here, we review some recent studies in which we have addressed several questionsregarding the short- and long-term effects of LP-induced OHT in the adult rodentretina: (i) What are the effects of OHT on the main retinorecipient target nuclei

in the brain? (ii) What are the main retrograde effects of OHT on the RGC tion? (iii) Does OHT affect other non-RGC neurons in the GCL? (iv) Does OHT

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popula-affect the outer retina? (v) What is the general response of ipRGCs to OHT-induced

retinal degeneration and BDNF afforded neuroprotection?

3.1 LP OF THE LIMBAL AND EPISCLERAL VEINS RESULTS IN OHT

The IOP in the nonlasered right eyes remained within normal levels throughout the

study for rats and mice In adult albino rats, LP resulted in significant IOP raises

dur-ing the first 24 h that reached peak values at around 48 h; these high levels were

maintained for the first week and then declined slowly to reach basal values by

3 weeks (Ortı´n-Martı´nez et al., 2015; Salinas-Navarro et al., 2010;

control values during the first 5 days, returned to basal levels at day 7, and remained so

for the rest of the study (Cuenca et al., 2010; Salinas-Navarro et al., 2009c;

smaller in the pigmented than in the albino mice (Valiente-Soriano et al., 2015a)

In our LP-induced OHT murine models, the IOP values raised considerably for

short periods of time, and this may be considered a disadvantage when compared

to more chronic models of OHT that result in a slower progression of RGC loss

Nevertheless, the IOP elevations obtained in our rat and mice studies result in a

num-ber of characteristic features such as sectorial RGC death, early damage to RGC

axons somewhere near the ON head, and survival of RGCs with their orthograde

and retrograde axonal transport impaired, all of which have been observed in the

DBA/2J inherited mouse model of glaucoma (Calkins, 2012; Crish et al., 2010;

Filippopoulos et al., 2006; Howell et al., 2007; Jakobs et al., 2005; Pe´rez de Lara

LP-induced OHT rodent models are not similar to monkey models of glaucoma

or OHT, learning from murine models may help our understanding of OHT-induced

retinal degeneration and contribute to design new strategies to treat and/or prevent

the disease progression

3.2 ANTEROGRADE EFFECTS OF OHT-INDUCED RETINAL

DEGENERATION ON THE RETINOTECTAL INNERVATION

Glaucoma is no longer considered a sole disease of the RGCs and their axons because

other structures of the primary visual pathway are affected (Yu¨cel et al., 2000, 2001,

2003), such as the main retinorecipient target nuclei in the brain that are responsible

for image-forming vision (Calkins, 2012; Crish and Calkins, 2011; Lambert et al.,

2011) Thus, it was important to investigate the effects of OHT on the innervation

of the visual layers of the SC, where adult rodent RGCs project massively (Perry,

ranging from 10 days to 14 weeks, the area and volume of the contralateral SC

occupied by retinal axon terminals identified with the CTB were analyzed The

la-beling of retinotectal axon terminals in control mice was homogenous throughout the

rostrocaudal extension of the SC, with an intense staining in the contralateral visual

9

3 Results and discussion

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layers of the SC and highest densities of CTB immunoreactivity in the stratum zonaleand stratum griseum superficiale, where RGC axons arborize, differentiate, and es-tablish synaptic contacts with target neurons In the experimental mice, there was amarked reduction in the amount of CTB-labeled retinal afferents in the superficiallayers of the contralateral SC; there were small patches in which CTB labelingwas reduced allowing observation of individual axons and their terminal arboriza-tions In addition, there were areas with little to none CTB immunoreactivity thatoften presented the form of a column extending in the dorsoventral axis of the visuallayers of the SC, resembling the deployment of rodent axon terminals (Ling et al.,

1998) and suggesting degeneration of retinal axons and their terminals, as observedfollowing other types of retinal insults (Avile´s-Trigueros et al., 2003; Mayor-Torroglosa et al., 2005; Vidal-Sanz et al., 2007) The lateral extension of these areasvaried from a small narrow column to almost one half or more of the SC mediolateralextension (Fig 1), whereas the rostrocaudal extension of the SC varied from a few toalmost 10–14 consecutive serial coronal sections Approximately 50% of the areaoccupied by the visual layers of the right SC did not show CTB-labeled retinal ter-minals (Fig 1) The amount of this lack of labeling did not progress between 10 daysand 14 weeks, and this is consistent with the observation that RGC loss in this miceOHT model does not progress during this period of time (Valiente-Soriano et al.,

retina and the diminution in retinotectal denervation In adult albino rats, Drouyer

structures with a range from approximately 50% in the ventral lateral geniculate cleus to 72% in the suprachiasmatic nucleus, and 50% in the SC Our results in adultpigmented mice are consistent with those found in adult albino rats and furtherstrengthen the idea that OHT results not only in marked degeneration of the RGClayer but also in the anterograde degeneration of retinofugal axons and thus in sig-nificant denervation of the retinorecipient target nuclei in the brain (Crish et al.,2010; Dekeyster et al., 2015; Yu¨cel et al., 2003)

nu-3.3 RETROGRADE EFFECTS OF OHT ON THE RGC POPULATION, NEUROPROTECTION WITH BDNF

In adult albino rats (Ortı´n-Martı´nez et al., 2015; Salinas-Navarro et al., 2010;

Valiente-Soriano et al., 2015a) mice, OHT resulted within the first 2 weeks in the loss of proximately 80% of the RGC population identified in the left (lasered) retinas withthe retrograde tracers FG or OHSt applied to both SCi 1 week prior to animal proces-sing These retinas showed areas that were almost devoid of retrogradely labeledRGCs and adopted the form of pie-shaped sectors with their base located on the ret-inal periphery and their apex toward on the optic disc; these areas were more frequent

ap-in the dorsal retap-inas and varied ap-in size from a small sector to one or several retap-inalquadrants In contrast, the right (control not lasered) retinas showed a normal

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distribution of RGCs (retrogradely labeled or immunostained with Brn3a) with

high-est densities in the visual streak, along the nasotemporal axis in the dorsal retina,

peaking in the superotemporal quadrant, as previously described (Nadal-Nicola´s

et al., 2009, 2012, 2014, 2015; Ortı´n-Martı´nez et al., 2010, 2014; Salinas-Navarro

of the topological distribution of surviving RGCs in these OHT retinas (Figs 2–4, 6,

and 8) We found variability in the severity of retinal damage, and this is in

agree-ment with previous reports from this (Vidal-Sanz et al., 2012) and other (Fu and

in the degree of degeneration has also been reported in an inherited pigmented mouse

FIGURE 1

Deafferentation of the contralateral superior colliculus after ocular hypertension (A) and

(B) Serial coronal brain sections spanning the right (contralateral) superior colliculus

(from anterior/posterior bregma coordinates:3.08 to 4.72 mm) showing retinal afferents

labeled by anterograde tracing with cholera toxin subunit B injected into a naı¨ve eye

(A) or a hypertensive left eye analyzed 14 weeks later (B)

11

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model of experimental glaucoma, the DBA/2J mice (Filippopoulos et al., 2006;Howell et al., 2007; Jakobs et al., 2005; Pe´rez de Lara et al., 2014; Schlamp

et al., 2006; Soto et al., 2008) In addition to this sectorial loss, the isodensity mapsalso revealed a diffuse loss, even within the retinal areas showing surviving RGCs.This amount of retinal degeneration was based on quantification of RGCs labeledwith retrograde tracers applied to the SCi 1 week prior to animal processing Whenthe surviving population of RGCs was identified with dextran tetramethylrhodamine(DTMR), a tracer that when applied to the ocular stump of the orbitally transected

ON diffuses passively toward the cell somata, or with Brn3a immunostaining, there

FIGURE 2

Ocular hypertension induces loss of orthotopic and displaced retinal ganglion cells Maps ofthree representative retinas (one per row) showing the distribution of retrogradely tracedorthotopic (oRGCs) (A, C, E) and displaced (dRGCs) (A0, C0, E0), and of Brn3a+oRGCs (B, D,F) or Brn3a+dRGCs (B0, D0, F0) in a naı¨ve rat (first row) or in experimental rats (secondand third row) 3 weeks after laser photocauterization of the limbar and episcleral vessels

to induce ocular hypertension The isodensity (C–F) and their corresponding neighbors(C0–F0) maps show a parallel topological loss between oRGCs and dRGCs (FG traced andBrn3a+), which is consistent with an axonal compression produced at the level of theoptic nerve head At the bottom of each map is shown the number of RGCs or dRGCsrepresented Color (different gray shades in the print version) scale for isodensity maps

in (B) bottom right, for neighbor maps in (A0) RE, right eye; LE, left eye; D, dorsal; V, ventral;

N, nasal; T, temporal Scale bar in (A)¼1 mm

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FIGURE 3 See legend on next page.

13

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was a clear mismatch between the numbers of traced RGCs and the numbers ofDTMR+RGCs or Brn3a+RGCs in the same retinas The numbers of Brn3a+RGCswere significantly greater than those of traced RGCs at early periods after LP butnot at surviving intervals of 5 weeks or more, indicating that at early time periodsfollowing OHT a large population of surviving RGCs had lost their active retrogradeaxonal transport (Agudo-Barriuso et al., 2013a; Vidal-Sanz et al., 2012); such an al-teration has been previously observed following other types of retinal or ON injuries

1 and 5 weeks after LP, the numbers of Brn3a+RGCs diminished significantly, cating that RGC loss was progressive between 1 and 5 weeks after LP

indi-It was interesting to observe that a single intraocular injection of 5mg of BDNFright after LP resulted in significant greater RGC survival when compared to vehicle-treated retinas examined at 12 or 15 days The numbers of Brn3a+RGCs in thevehicle-treated retinas examined at 12 or 15 days represented 56% (n¼4) or 45%(n¼4), respectively, of the control values, whereas for the BDNF-treated retinasthese proportions were 83% (n¼4) or 72% (n¼4) at 12 or 15 days, respectively(Valiente-Soriano et al., 2015b) These findings are consistent with previous studies(Fu et al., 2009; Ko et al., 2001; Martin et al., 2003; Quigley et al., 2000; Wilson and

glial-derived neurotrophic factor to afford transient neuroprotection against induced RGC death (Di Polo et al., 1998; Galindo-Romero et al., 2013b;Lindqvist et al., 2004; Parrilla-Reverter et al., 2009a; Sa´nchez-Migallo´n et al., 2011).The nerve fiber layer of the retina was investigated at periods of time rangingfrom 1 to 12 weeks after OHT (Salinas-Navarro et al., 2009c, 2010; Vidal-Sanz

of the heaviest neurofilament subunit (Garcı´a-Ayuso et al., 2014; Marco-Gomariz

FIGURE 3

The loss after OHT is selective to RGCs in the GCL Isodensity maps from a representativeexperimental retina 15 days after laser photocauterization of the perilimbar and episcleralveins, immunoreacted for Brn3a (A) and stained with DAPI in the ganglion cell layer (B).The Brn3a isodensity map shows a typical pie-shaped retinal sectors lacking RGCs in anexperimental retina 15 days after LP-induced OHT The same retina shows large numbers

of DAPI-stained nuclei in the areas lacking Brn3a+RGCs as reflected in the DAPI

isodensity map (B) Bottom of each map: number of cells counted in that retina Densitycolor (different gray shades in the print version) scale in A and B bottom right ranges from

0 (purple (black in the print version)) to3500 RGCs/mm2

or5000 DAPI+

nuclei (red(gray in the print version)), respectively (C–E) Higher power micrographs from the inset

in A, B showing Brn3+RGCs (C), calretinin+neurons (D), and DAPI+nuclei (E) to illustratethat in the retinal sectors with diminished numbers of Brn3a+RGCs there were largenumbers of DAPI+nuclei (E) many of which are displaced amacrine cells (calretinin+neurons,D) in the GCL LE, left eye; D, dorsal; V, ventral; N, nasal; T, temporal Scale bar for(A) and (B)¼1 mm Scale bar for (C–D)¼50 mm

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et al., 2006; Parrilla-Reverter et al., 2009b; Villegas-Pe´rez et al., 1996) There were

abnormal RT97 staining in bundles of axons and RGCs that were mainly located

out-side the areas of the retina containing surviving RGCs This abnormal RT97 staining

consisted of axonal beadings and varicosities as well as intense staining of the RGC

somata, all of which are typically observed after ON axotomy (Parrilla-Reverter

FIGURE 4

Normal appearance of retinal vessels in ocular hypertensive retinas (A, A0) Naı¨ve retina

retrogradely labeled with fluorogold (FG) applied to both superior colliculi 1 week prior to

animal processing and its corresponding isodensity map (B) The retinal vessels

immunostained with RECA1 antibodies in a black and white wholemount retinal

reconstruction (C, D) Details of the retina (A), taken from the dorsotemporal (C) and

inferotemporal (D) quadrant showing FG+RGCs (white), Brn3a+RGCs (red (black in the

print version)), and RECA1+vessels (green (gray in the print version)) In the naı¨ve retina,

there is competent retrograde axonal transport (RAT) and the immunostained retinal

vessels appear normal Two weeks after laser photocauterization of the perilimbal and

episcleral vessels, an ocular hypertensive retina shows typical loss of the RAT in the

dorsal retina along a large sector spanning from 8 to 5 o0clock (E–E0) The retinal vessels,

in the black and white representation (F), appear normal and morphologically similar to

the control naı¨ve retina These are also observed in the magnification taken from an area

with no RAT (G) or with RAT (H) D, dorsal; V, ventral; T, temporal; N, nasal

15

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et al., 2009b; Vidal-Sanz et al., 1987; Villegas-Pe´rez et al., 1988) Interestingly,within the areas of the retina lacking retrogradely labeled RGCs, there wereRT97+RGCs as well as bundles of RT97+axons These bundles of RT97+axons wereobserved up to 12 weeks after OHT at a time when the vast majority of the RGCpopulation is already disconnected from their target, suggesting that retrograde de-generation of the intraretinal axon is a lengthy process (Buckingham et al., 2008;Howell et al., 2007; Salinas-Navarro et al., 2010; Schlamp et al., 2006; Soto

Parrilla-Reverter et al., 2009b), it is tempting to suggest that it is difficult to predict RGCsurvival based on the appearance of the nerve fiber layer of the retina, since theappearance of intraretinal axons does not mirror the population of RGCs connected

to the brain (Vidal-Sanz et al., 2012)

3.4 OHT AFFECTS SELECTIVELY THE RGC POPULATION IN THE GCL

Several groups of mouse and rat OHT retinas were analyzed in wholemounts toinvestigate the fate of neurons in the GCL of the retina In addition to the RGCpopulation, within the GCL there is a population of displaced amacrine cells asnumerous as the population of RGC itself (Perry, 1981; Perry and Cowey, 1979;Schlamp et al., 2013) In our studies in albino rats (Ortı´n-Martı´nez et al., 2015)and pigmented mice (Valiente-Soriano et al., 2015a), it is likely that most of theDAPI+nuclei observed in the pie-shaped retinal sectors lacking Brn3a+RGCs are ac-tually displaced amacrine cells (Fig 3), although a small proportion may correspond

to endothelial cells, astrocytes, and microglia which are known to respond withproliferation or cell migration (Rojas et al., 2014; Salvador-Silva et al., 2000;

in the sectorial RGC loss, the inner retinal vessels were immunolabeled with RECA1and examined in wholemounts There were no apparent vascular abnormalities in theregions that showed FG+or Brn3a+RGCs nor in regions lacking RGCs that couldaccount for the sectorial loss of RGCs (Fig 4; Valiente-Soriano et al., 2015b) Ifthe mechanism leading to OHT-induced retinal degeneration were to act directly

in the retina, neurons in the GCL should be affected, but this was not so The ing observations: (i) the typical pie-shaped sectors lacking axons and their parentRGCs, including the small subpopulation of displaced RGCs (Fig 2); (ii) the pres-ence in those pie-shaped sectors of non-RGC neurons, as observed with DAPIand calretinin staining (Fig 3), presumably displaced amacrine cells; and (iii) thepreservation of the normal appearance of the inner retinal vasculature (Fig 4), are inagreement with previous studies (Cone et al., 2010; Jakobs et al., 2005; Kielczewski

population in the GCL, and overall speak in favor of some type of mechanicalcompression-like damage to bundles of axons in the ON head While the mechanismsunderlying ON injury in glaucoma are not fully understood, among the main lines ofthought are the mechanical and vascular theories The mechanical theory argues thatthe pressure at the level of the ON head would result in direct compression of bundles

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of axons (Morgan et al., 1988) thus inducing an axotomy-like insult that would result

in an orthograde as well as in a protracted retrograde degeneration of the intraretinal

portion of the axons and their parent cell somata The mechanical theory would

ex-plain the typical pie-shaped loss of RGCs and their axons that is observed in OHT

models in rats and mice Elevated IOP has been shown to cause posterior

deforma-tion of the ON head, which is more pronounced on the dorsal than in the ventral

as-pect, thus providing a plausible explanation for the greater susceptibility to damage

in the dorsal retina (Fortune et al., 2011) In addition to this typical sectorial loss, the

observation of diffuse loss could well be explained because such a constriction of

axons could also affect ON fibers within the main ON head that are somehow more

susceptible to pressure-induced injury The vascular theory implies compression of

capillaries that supply the ON head (Pillunat et al., 1997), and this could result in a

compromise of the vascular supply to ON fibers within the ON head Additional

mechanisms may involve the lack of functional properties of astrocytes at the level

of the ON head (Dai et al., 2012; Nguyen et al., 2011)

3.5 OHT AFFECTS THE ORLs

Recent studies from this laboratory on OHT-induced retinal degeneration in adult

rats and mice have shown that in addition to severe RGC loss, there are also

impor-tant functional and structural alterations of the ORLs These studies employed

molecular (Cuenca et al., 2010; Ortı´n-Martı´nez et al., 2015), functional (

and retinal wholemounts (Cuenca et al., 2010; Ortı´n-Martı´nez et al., 2015;

S- and L-opsin mRNA expression diminished significantly in OHT retinas analyzed

15 days to 3 months after OHT when approximately 20% of the original protein

levels were quantified (Ortı´n-Martı´nez et al., 2015), and these data are in agreement

with previous reports in adult rats (Drouyer et al., 2008) and monkeys with

exper-imental OHT and in human eyes from glaucomatous donors (Pelzel et al., 2006)

The effects of increased IOP on the outer retina were investigated in radial sections

of the retina in adult Swiss mice and albino rats at various time intervals ranging from

3 to 14 weeks in mice (Cuenca et al., 2010) and from 1 to 6 months in rats (

Ortı´n-Martı´nez et al., 2015) In adult Swiss mice, at 3 months there were significant

dim-inutions in the thickness of the outer nuclear and plexiform layer when compared to

their fellow eyes (Cuenca et al., 2010) In adult rats, 6 months after LP, the OHT

retinas showed multiple small areas of degeneration in the outermost (outer nuclear

and outer segment) retinal layers (Fig 5), and the morphometric analysis showed

significant reductions (30%) in the OHT retinas when compared to the control values

for the outer nuclear layer (Ortı´n-Martı´nez et al., 2015) The flash electroretinogram

(ERG), scotopic threshold response (STR), and a- and b-wave amplitudes were

recorded before and at various times after OHT induction in adult Swiss mice

the STR and a- and b-waves of the ERG that appeared as early as 24 h after LP

17

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and did not recover throughout the 2- to 3-month period of study indicating that creased IOP results in severe damage to the innermost, inner nuclear, and outer nu-clear layers of the retina (Abbott et al., 2014; Cuenca et al., 2010; Salinas-Navarro

15 days and 6 months to determine the total numbers of L- and S-cones (Fig 6).When compared to a group of sham experiments, there were significant losses ofL- and S-cones that progressed up to 6 months and amounted to 19% and 33% ofthe original population by 1 month, to 62% and 51% by 3 months, and to 66%and 59% by 6 months, respectively (Fig 7) The isodensity maps showed a lack

of L- and S-cone immunoreactivity that was diffuse throughout the retina but alsolocalized in patchy areas of the retina (Fig 6) These focal areas of the retina lackingboth types of L- and S-cone immunoreactive outer segments may well correspond tothe focal regions of outer layer degeneration observed in radially oriented paraffin-embedded cross sections illustrated inFig 5 To ascertain if L- and S-cone loss wasrelated to RGC degeneration, we compared the distribution of RGCs and L- andS-cones in the same retinas and found that the geographical pattern of RGC losswas not related to that of L- and S-cones and vice versa In addition, the effects

of ON transection, an injury that induces massive loss of RGCs (Villegas-Pe´rez

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the populations of L- and S-cones were normal Taken together, these experiments

indicate that cone loss appears to be independent of RGC loss (Ortı´n-Martı´nez

et al., 2015)

Overall, our findings using molecular (Cuenca et al., 2010; Ortı´n-Martı´nez et al.,

2015), functional (Salinas-Navarro et al., 2009c), and morphological (Cuenca et al.,

that following OHT, there is an important degeneration of the outer nuclear and outer

segment retinal layers Our results are in agreement with previous reports showing

that OHT also results in adverse effects on the cone photoreceptor population: (i) in

rodent models analyzed with morphometric techniques (Abbott et al., 2014;

Ferna´ndez-Sa´nchez et al., 2014; Fuchs et al., 2012; Guo et al., 2010; Rojas et al.,

2014), with functional techniques (Chen et al., 2015; Georgiou et al., 2014;

Heiduschka et al., 2010; Mittag et al., 2000; Pe´rez de Lara et al., 2014), or with both

FIGURE 6

Topological analysis of RGCs, L-cones, and S-cones 3 months after OHT Isodensity maps

showing topological distributions of entire populations of retinal ganglion cells (RGCs),

L-cones, or S-cones in the same retinas Top row corresponds to a control right retina and

bottom row shows a representative left experimental retina 3 months after lasering the

perilimbar and episcleral veins to induce ocular hypertension (OHT) RGCs were identified

with fluorogold applied to both superior colliculi 1 week prior to animal processing L- and

S-cones were immunodetected with antibodies against L- and S-opsin, respectively Color

(different gray shades in the print version) scale range: RGCs/mm2goes from 0 (purple (black

in the print version)) to 2500 or higher (red (gray in the print version)); L-cones/mm2, from

0 (purple (black in the print version)) to 6500 or higher (red (gray in the print version));

S-cones/mm2, from 0 (purple (black in the print version)) to 1300 or higher (red (gray in the

print version)) S, superior; T, temporal; I, inferior; N, nasal Scale bar: 1 mm

19

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(Bayer et al., 2001; Calkins, 2012); (ii) in monkey OHT models analyzed with atomical techniques (Nork, 2000; Nork et al., 2000) or with functional techniques

func-tional (Barboni et al., 2011; Bessler et al., 2010; Drasdo et al., 2001; Holopigian

et al., 1990; Kanis et al., 2010; Niwa et al., 2014; Velten et al., 2001; Werner

et al., 2011) or anatomical (Choi et al., 2011; Kanis et al., 2010; Lei et al., 2008,

techniques The above reviewed results appear to be in contrast with previous studiesthat have not found cone photoreceptor loss in OHT monkey (Wygnanski et al.,

1995) or human (Kendell et al., 1995) retinas Several explanations may accountfor these discrepancies Our results underscore that only long-term studies may re-veal the effects of OHT, while most of the previous reports are short-term studies,and this might explain why they did not find evidence of photoreceptor or any othernon-RGC retinal damage Our OHT rodent models may resemble an acute angle-closure human glaucoma coursing with elevated IOP peaks (Vidal-Sanz et al.,

2012), and these increments mimic those reported to be associated with cone loss

that the levels of IOP reached in this model may have induced choroidal ciency that results in outer retinal pathology (Nork et al., 2014) Nevertheless, our

up to 6 months, except for S-cones between 3 and 6 months and for L-cones between

15 days and 1 month Data for the values in sham and OHT at 15 days, and 1 or 6months were obtained fromOrtı´n-Martı´nez et al (2015)and values in OHT at 3 monthswere obtained from a group prepared for the present study

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OHT models resulted in sectorial loss of RGCs and degeneration of the nerve fiber

layer that were also found by others (Fu and Sretavan, 2010), and in the DBA/2J mice

model of OHT (Buckingham et al., 2008; Jakobs et al., 2005; Schlamp et al., 2006;

Soto et al., 2008), thus arguing in favor of a similar noxius stimuli In our studies, we

have examined the entire retina in wholemounts rather than sampling a few areas of

the retina, and this is a better approach to investigate the populations of L- and

S-cones Thus, overall our studies suggest that photoreceptor loss may constitute

an important feature of the retinal pathology associated with OHT and this may

be-come relevant to human glaucoma (Ortı´n-Martı´nez et al., 2015) Thus, it is likely that

the concept of glaucoma as a solely disease of the retina and ON may evolve to

broader implications of the primary visual pathway, including the outer retina

3.6 THE MELANOPSIN RGC POPULATION RESPONDS DIFFERENTLY

TO OHT-INDUCED RETINAL INJURY

Several studies have suggested that m+RGCs survive better to a number of noxious

stimuli (Cui et al., 2015) including OHT (Li et al., 2006) Our recent studies in ocular

hypertensive rat and mouse retinas indicate that m+RGC survival was similar to the

rest of the main RGC population (identified with Brn3a antibodies), at least for OHT

retinas that were examined 2–12 weeks after lasering the episcleral and perilimbal

vessels RGC survival after 2 weeks of LP was roughly 50% for the Brn3a+RGCs

or m+RGCs, both in pigmented mice (Valiente-Soriano et al., 2015a) and albino rats

resistant to ON axotomy (Pe´rez de Sevilla Mu¨ller et al., 2014; Robinson and

and inherited optic neuropathies (La Morgia et al., 2010), it is not clear yet if they

are also more resistant to OHT-induced retinal injury (de Zavalı´a et al., 2011;

El-Danaf and Huberman, 2015; Jakobs et al., 2005; Li et al., 2006; Wang et al.,

when compared to the general population of RGCs, the responses of m+RGCs to

OHT-induced retinal degeneration differed in two important aspects: (i) the

distribu-tion of surviving m+RGCs within the retinas did not show the typical sectorial pattern

observed for the general population of Brn3a+RGCs, but rather a diffuse pattern of

m+RGCs loss (Fig 8) and (ii) m+RGCs were not responsive to BDNF-induced

neu-roprotection, while non-m+RGCs (identified with Brn3a) in the same retinas showed

a significant greater survival (Valiente-Soriano et al., 2015b) The typical sectorial

RGC and axonal loss found in the rodent OHT retinas is currently explained by an

axotomy-like insult to bundles of axons somewhere near the ON head where

retino-topic arrangement is maximal (Fitzgibbon and Taylor, 1996; Guillery et al., 1995;

Jeffery, 2001; Jeffery et al., 2008; Vidal-Sanz et al., 2012) The absence of a sectorial

loss in m+RGCs might be explained either by one of the following possibilities;

m+RGCs might have an intraretinal axon collateral (Joo et al., 2013; Semo et al.,

2014) that could provide trophic support when the main retinofugal axon results

damaged, or m+RGCs lack retinotopy Retinal innervation of nonimage-forming

21

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FIGURE 8

Topology of Brn3a+RGCs and m+RGCs in naı¨ve and hypertensive retinas (A–A0) Isodensitymaps illustrating the topological distribution of the Brn3a+RGC population (A) and thecorresponding neighbor map of the melanopsin-expressing (m+RGC) population (A0) in

a naı¨ve retina Ocular hypertensive retinas analyzed 15 days (B) or 3 months (C) after laserphotocauterization of the perilimbar and episcleral vessels to induce ocular hypertension show

in their isodensity maps of Brn3a+RGCs, both sectorial and diffuse damage in each retina.The neighboring maps representing m+RGCs (B0, C0) show their diffuse loss throughoutthe retina although this is more severe in the dorsotemporal retina At the bottom left ofeach map is shown the number of Brn3a+RGCs or m+RGCs represented Color (differentgray shades in the print version) scale for isodensity maps is shown in (A) and rangesfrom 0 (purple (black in the print version)) to2500 (red (gray in the print version)) RGCs/mm2.Color (different gray shades in the print version) scale for the neighbor map is shown in (A0)and ranges from 0–2 (purple (black in the print version)) to21–23 (red (gray in theprint version)) neighbors in a radius of 0.22 mm S, superior; I, inferior; T, temporal; N, nasal

Trang 32

visual centers is known to lack retinotopy, and such a lack of retinotopic arrangement

for the m+RGC axons at the level of the ON head could explain the diffuse pattern

rather than the sectorial pattern of cell loss

The present studies rely on immunostaining several proteins to identify different

ret-inal neurons, and thus caution should be taken when interpreting the results because

retinal injury may modify the expression of many genes (Agudo et al., 2008, 2009;

Agudo-Barriuso et al., 2013b; Chidlow et al., 2005); this may result in lower

expres-sion of the proteins and their epitopes Nevertheless, both melanopsin and Brn3a

have been shown to be expressed long after ON injury (Galindo-Romero et al.,

2013b; Sa´nchez-Migallo´n et al., 2011)

Our results indicate that following LP: (i) the SC shows that the volume occupied

by retinotectal afferents is reduced to approximately half their normal values;

(ii) within the GCL of the retina, OHT affects selectively projecting neurons, that

is, RGCs, but not displaced amacrine cells; (iii) intraocular administration of BDNF

resulted in significantly increased RGC survival at 12 and 15 days after OHT; (iv) the

inner retinal vessels did not show abnormalities that could be responsible for the

sec-torial loss of RGCs; (v) m+RGCs are numerically affected in the same proportion as

the rest of RGCs but show important differences; their topographic loss is diffuse and

not sectorial, and does not respond to BDNF neuroprotection; and (vi) with time

there is progressive functional and morphological alterations of the outermost retinal

layers with severe loss of S- and L-cones, and such a time course progression

indi-cates that outer retinal pathology does not reverse when OHT disappears (by 3 weeks

after LP), but on the contrary continues progressing for long periods of time up to

6 months

ACKNOWLEDGMENTS

We are grateful to our collaborators that have contributed to the various studies in the

Laboratory of Experimental Ophthalmology over the years The technical help of Leticia

Nieto-Lo´pez and Jose´ Manuel Bernal-Garro is greatly acknowledged

Financial support for these studies was obtained from Spanish Ministry of Economy and

Competitiveness: SAF-2012-38328; ISCIII-FEDER “Una manera de hacer Europa”

PI13/01266, PI13/00643, RETICS: RD12/0034/0014

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