Progress in brain research, volume 220

Serial Editor

Vincent Walsh
Institute of Cognitive Neuroscience
University College London
17 Queen Square
London WC1N 3AR UK

Editorial Board
Mark Bear, Cambridge, USA.
Medicine & Translational Neuroscience
Hamed Ekhtiari, Tehran, Iran.
Addiction
Hajime Hirase, Wako, Japan.
Neuronal Microcircuitry
Freda Miller, Toronto, Canada.
Developmental Neurobiology
Shane O’Mara, Dublin, Ireland.
Systems Neuroscience
Susan Rossell, Swinburne, Australia.
Clinical Psychology & Neuropsychiatry
Nathalie Rouach, Paris, France.
Neuroglia
Barbara Sahakian, Cambridge, UK.
Cognition & Neuroethics
Bettina Studer, Dusseldorf, Germany.
Neurorehabilitation
Xiao-Jing Wang, New York, USA.
Computational Neuroscience

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Contributors
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
Departamento de Oftalmologı´a, Universidad de Murcia and Instituto Murciano de
Investigacio´n Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
Marcelino Avile´s-Trigueros
Departamento de Oftalmologı´a, Universidad de Murcia and Instituto Murciano de
Investigacio´n Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
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


vi

Contributors

Filippo Drago
Department of Biomedical and Biotechnological Sciences, Section of
Pharmacology, University of Catania, Catania, Italy
Stefano Forte
IOM Ricerca srl, Catania, Italy
Beatriz I. Gallego
Instituto de Investigaciones Oftalmolo´gicas Ramo´n Castroviejo, Universidad
Complutense de Madrid, Spain
Roberto Gallego-Pinazo
Ophthalmic Research Unit “Santiago Grisolı´a”, University Hospital Dr. Peset, and
Department of Ophthalmology, University and Polytechnic Hospital la Fe,
Valencia, Spain

Diego Garcı´a-Ayuso
Departamento de Oftalmologı´a, Universidad de Murcia and Instituto Murciano de
Investigacio´n Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
Jose´ J. Garcı´a-Medina
Ophthalmic Research Unit “Santiago Grisolı´a”, University Hospital Dr. Peset,
Valencia; Department of Ophthalmology, University Hospital Reina Sofia, and
Department of Ophthalmology and Optometry, University of Murcia, Murcia,
Spain
Neeru Gupta
Department of Ophthalmology and Vision Sciences; Department of Laboratory
Medicine and Pathobiology, University of Toronto; Keenan Research Centre for
Biomedical Science, and Glaucoma and Nerve Protection Unit, St. Michael’s
Hospital, Toronto, ON, Canada
Manuel Jime´nez-Lo´pez
Departamento de Oftalmologı´a, Universidad de Murcia and Instituto Murciano de
Investigacio´n Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
Nele Kindt
Amakem Therapeutics, Diepenbeek, Belgium
Hani Levkovitch-Verbin
Glaucoma Service, Goldschleger Eye Institute, Sheba Medical Center, and
Sackler Faculty of Medicine, Tel-Aviv University, Tel-Hashomer, Israel
Fumihiko Mabuchi
Department of Ophthalmology, Faculty of Medicine, University of Yamanashi,
Chuo, Yamanashi, Japan
Keith Martin
John van Geest Centre for Brain Repair, University of Cambridge; Cambridge
NIHR Biomedical Research Centre, and Wellcome Trust Medical Research
Council Cambridge Stem Cell Institute, Cambridge, UK

Contributors

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
Departamento de Oftalmologı´a, Universidad de Murcia and Instituto Murciano de
Investigacio´n Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
Carlo Nucci
Ophthalmology Unit, Department of Experimental Medicine and Surgery,
University of Rome Tor Vergata, Rome, Italy
Arturo Ortı´n-Martı´nez
Departamento de Oftalmologı´a, Universidad de Murcia and Instituto Murciano de
Investigacio´n Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
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
Department of Biomedical and Biotechnological Sciences, Section of
Pharmacology, University of Catania, Catania, Italy
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

vii


viii

Contributors

Blanca Rojas
Instituto de Investigaciones Oftalmolo´gicas Ramo´n Castroviejo, Departamento de
Oftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,
Spain
Giovanni Luca Romano
Department of Biomedical and Biotechnological Sciences, Section of
Pharmacology, University of Catania, Catania, Italy
Laura Rombola`

Department of Pharmacy, Health and Nutritional Sciences, Section of Preclinical
and Translational Pharmacology, University of Calabria, Cosenza, Italy
Rossella Russo
Department of Pharmacy, Health and Nutritional Sciences, Section of Preclinical
and Translational Pharmacology, University of Calabria, Cosenza, Italy
Yoichi Sakurada
Department of Ophthalmology, Faculty of Medicine, University of Yamanashi,
Chuo, Yamanashi, Japan
Juan J. Salazar
Instituto de Investigaciones Oftalmolo´gicas Ramo´n Castroviejo, Facultad de
O´ptica y Optometrı´a, Universidad Complutense de Madrid, Spain
Manuel Salinas-Navarro
Departamento de Oftalmologı´a, Universidad de Murcia and Instituto Murciano de
Investigacio´n Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
Elena Salobrar-Garcı´a
Instituto de Investigaciones Oftalmolo´gicas Ramo´n Castroviejo, Universidad
Complutense de Madrid, Spain
Salvatore Salomone
Department of Biomedical and Biotechnological Sciences, Section of
Pharmacology, University of Catania, Catania, Italy
Ingeborg Stalmans
Laboratory of Ophthalmology, KU Leuven, and Department of Ophthalmology,
University Hospitals Leuven (UZ Leuven), Leuven, Belgium
Nicholas Strouthidis
NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation
Trust and UCL Institute of Ophthalmology, London, UK; Discipline of Clinical
Ophthalmology and Eye Health, University of Sydney, Sydney, NSW, Australia,
and Singapore Eye Research Institute, Singapore, Singapore
Alberto Trivin˜o
Instituto de Investigaciones Oftalmolo´gicas Ramo´n Castroviejo, Departamento de

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


Contributors

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

ix


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 typical 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 pathophysiology 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 tricarboxylic 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 antioxidant capacity has been reported along with increased end-products of lipid peroxidation, 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 clinical proof.
Experimental data indicate that axonal injury triggers rapid structural alterations
in RGC dendritic arbors, prior to manifest axonal loss, leading to synaptic rearrangements and functional deficits.
Tissue remodeling occurring in glaucoma may cause biomechanical and microstructural changes that are likely to alter the mechanical environment of the optic
nerve head and may contribute to axonal damage. Indeed, experimental evidence following 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 morphological 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

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xx

Preface: New trends in basic and clinical research of glaucoma

of the retinal macroglia and microglia in the uninjured eye along with important observations implicating innate and adaptive immunity. The latter data support a role
for blood–retina barrier disruption in the pathophysiology of glaucoma-associated
neurodegenerative process other than simply suggesting that the eye contralateral
to experimental glaucoma cannot be a true control.
Experimental data do support the hypothesis that autophagy might participate in
the process leading to RGC death though the precise role awaits to be clarified. In
fact, evidence shows that downregulation of autophagy-related genes (Atg5, Atg7,
and BECN1) in normal human aging brain has been reported. On the contrary, a recent study analyzing LC3 and p62 levels in fresh TM from human donors reported
lower levels of p62 and increased LC3II/LC3I ratio in subjects older than 60 years
suggesting 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 compromised cells has been recently noticed. Accordingly, age-related dysfunction of
autophagy in the retina might represent another determinant for glaucoma
progression.
Indeed, association of glaucoma with age-related neurodegenerative diseases
stems from these sharing similar miRNAs regulated transduction pathways (see also

Part B for additional evidence). In fact, by means of in silico approaches and access
to bioinformatic resources, deregulated miRNAs in glaucoma, in age-related macular degeneration (AMD) and Alzheimer’s disease (AD), respectively, have been
found. Actually, 88 predicted miRNAs are common to glaucoma and AMD;
19 are common to glaucoma and AD; and 9 are common to AMD and AD. These
findings provide a valuable hint to assess deregulation of specific miRNA as potential biomarkers and therapeutic targets, in glaucoma and other neurodegenerative
diseases by means of preclinical and clinical studies.
The wealth of the above-mentioned data in conjunction with important news
emerging from clinical genetics and cell therapy technology is deeply discussed
by authoritative, world-widely recognized, scientists in this issue (Part A) of
Progress in Brain Research dedicated to glaucoma. To them is addressed our sincere
acknowledgment for making the issue a success. Also, our thanks go to the skillful
technical collaboration of individuals belonging to the Production Department of
Elsevier. We are especially indebted to Shellie Bryant for her continuous and highly
qualified editorial assistance from the very beginning of this venture.
The Editors
Giacinto Bagetta and Carlo Nucci


CHAPTER

Retinal neurodegeneration in
experimental glaucoma

1

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 Alarco´n-Martı´nez, Diego Garcı´a-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:

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 axonal transport, melanopsin immunodetection to identify intrinsically photosensitive RGCs
(m+RGC), Brn3a immunodetection to identify most RGCs but not m+RGCs, RECA1 immunodetection 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 morphometrically 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 diminished 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, />© 2015 Elsevier B.V. All rights reserved.

1

2

CHAPTER 1 Ocular hypertension-induced retinal degeneration

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

1 INTRODUCTION
The progressive loss of retinal ganglion cells (RGCs) and their axons with concomitant insidious defects in the visual field has been the classic hallmark of the glaucomatous optic neuropathies (GONs) (Chauhan et al., 2014; Quigley, 2011;
Weinreb et al., 2014). This concept however has evolved, and it is now well established that GONs involve not only the RGC population, the nerve fiber layer of the
retina (O’Leary et al., 2012), the optic disc, and optic nerve (ON) head (Chauhan
et al., 2009), but also the main retinorecipient subcortical and cortical nuclei of
the primary visual pathway, such as the lateral geniculate nucleus and primary
and 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
et al., 2003). Moreover, other nonvisually related areas of the cortex may also become 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 visual 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), the
administration 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
(Levkovitch-Verbin et al., 2002; WoldeMussie et al., 2001). In addition, there are
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 pigmentary glaucoma (Buckingham et al., 2008; Danias et al., 2003; Filippopoulos
et al., 2006; Panagis et al., 2010; Pe´rez de Lara et al., 2014; Reichstein et al.,
2007). In our laboratory, laser photocoagulation (LP) of the limbar tissues has
been 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.,
2009; Valiente-Soriano et al., 2015b) and in albino (Cuenca et al., 2010; de Hoz
et al., 2013; Dekeyster et al., 2015; Gallego et al., 2012; Rojas et al., 2014;


1 Introduction

Salinas-Navarro et al., 2009c) or pigmented (Nguyen et al., 2011; Valiente-Soriano
et al., 2015a) mice. In the LP–OHT models, typical observations are a sectorial loss
of RGCs, an initial damage to RGC axons somewhere near the ON head, and an alteration 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 neuroprotective agents ( Jehle et al., 2008; Vidal-Sanz et al., 2000, 2001, 2007), including 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;

Sa´nchez-Migallo´n et al., 2011). Indeed, the administration of BDNF has been shown
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
et al., 2000; Wilson and Di Polo, 2012). Here, we review some recent studies on
the neuroprotective effects of BDNF on the population of injured RGCs, including
melanopsin-expressing (m+RGCs) and nonmelanopsin-expressing RGCs (ValienteSoriano 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 glaucoma 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;
Ortı´n-Martı´nez et al., 2015; Salinas-Navarro et al., 2009a). These changes range
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
(El-Danaf and Huberman, 2015), most of the responses of RGCs to OHT-induced
retinal degeneration have been studied as a whole. Intrinsically photosensitive
RGCs (ipRGCs) mediate a number of nonimage-forming visual functions such as

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4

CHAPTER 1 Ocular hypertension-induced retinal degeneration

photoentrainment of the circadian rhythms, photic suppression of melatonin secretion, and pupillary light reflexes (Berson et al., 2002; Hankins et al., 2008; Hattar
et al., 2002; Semo et al., 2010; Vugler et al., 2015), and express the photopigment
melanopsin which can be readily identified with melanopsin antibodies (m+RGCs).
This tool provides unique opportunities to examine the responses of one of the many
types 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, respectively; 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.,
2014; Vugler et al., 2015), and recent evidence indicates glaucoma courses with a
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). Moreover, experimental glaucoma in rats has been shown to present important alterations
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 to
intraocular administration of BDNF.
In the following lines, we review some recent studies in our laboratory on rat and
mice 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). Using
modern techniques to identify and map in the same retinal wholemounts, the general
population of RGCs (nonmelanopsin expressing, identified with Brn3a antibodies),
the population of m+RGCs, the population of calretinin-expressing displaced amacrine cells, the subpopulation of displaced RGCs, the entire cell population in the
ganglion cell layer (GCL) (identified with DAPI nuclear staining), the nerve fiber
layer of the retina (identified with neurofilament antibodies), and the inner arterial
retinal vasculature (identified with RECA1 immunostaining), we have investigated
the responses of non-m+RGCs to OHT-induced retinal degeneration and neuroprotection afforded by BDNF and compared them to those of m+RGCs. Moreover, we
have examined up to 6 months the effects of OHT on the ORLs in radial cross sections of the retinas as well as in wholemounts, in which we have quantified and
mapped the populations of surviving RGCs, S- and L-cones. Finally, using cholera
toxin B subunit (CTB) as a fine anterograde tracer, we have investigated the fate of

the retinal terminals in their main target in the brain, the contralateral SC.

2 METHODS
2.1 ANIMAL HANDLING
Experiments were prepared in accordance with the ARVO, the European Union
guidelines for the use of animals in research, and the Ethical and Animal Studies
Committee of the University of Murcia (UM). Adult female albino Sprague–Dawley


2 Methods

(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 water ad 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
et al., 2013; Gallego et al., 2012; L€
onngren et al., 2006; Ramı´rez et al., 2010;
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; Quantel Medical, Clermont-Ferrand, France) of the perilimbal and episcleral vessels
(Cuenca et al., 2010; Levkovitch-Verbin et al., 2002; Salinas-Navarro et al.,

2009c, 2010; Vidal-Sanz et al., 2012; WoldeMussie et al., 2001). IOP was monitored
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) (SalinasNavarro et al., 2009c). With the exception of the readings taken at 12 h after LP,
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 methanesulfonate (OHSt; Molecular Probes, Leiden, The Netherlands) which is a small molecule 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, 5 mg 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.,
2015b). To identify the retinofugal projection, 4 days before sacrifice, 2.5 ml of
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


6

CHAPTER 1 Ocular hypertension-induced retinal degeneration

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 the
retinal orientation by making four radial cuts (the deepest in the superior pole) as
previously 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 (30 mm thick) from the level of the anterior thalamus to
the rostral pole of the cerebellum were obtained on a freezing cryostate (Avile´sTrigueros et al., 2000).

2.3.3 Retinal Cross Sections
Eyes were embedded in paraffin (Garcı´a-Ayuso et al., 2010; Ortı´n-Martı´nez et al.,
2015), and 3-mm-thick cross section cut in the parasagittal plane comprising the
superior and the inferior retina within the width of the ON head was obtained in a
microtome (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 out
following 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;
Valiente-Soriano et al., 2014, 2015a; Wang et al., 2003). L- and S-cones were double
immunodetected by their specific opsin expression (Garcı´a-Ayuso et al., 2013;
Ortı´n-Martı´nez et al., 2010, 2014). All RGCs (except m+RGCs) were immunodetected with Brn3a (Galindo-Romero et al., 2011; Nadal-Nicola´s et al., 2009,
2012, 2014, 2015), m+RGCs were detected with melanopsin (Galindo-Romero
et al., 2013a; Nadal-Nicola´s et al., 2014; Valiente-Soriano et al., 2014, 2015a,b), displaced amacrine cells with calretinin (Ortı´n-Martı´nez et al., 2015), and inner retinal
vessels with RECA1 (Valiente-Soriano et al., 2015b). To study all cells in the GCL,

retinal wholemounts were stained with DAPI (Vectashield mounting medium with
DAPI; Vector Laboratories, Inc., Burlingame, CA). For details about the primary
antibodies employed in this study, see Table 1.
Secondary fluorescent antibodies were donkey anti-goat Alexa Fluor 594, donkey 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 immunolocalized
with goat anti-CTB antibody and the ABC complex immunoperoxidase method
(Vectastain ABC Kit Elite; Vector Laboratories, Burlingame, CA) as previously


2 Methods

Table 1 Primary antibodies used in this work
Detection
of

Antigen

Antibody

Source

Retinotectal
terminals

Cholera toxin
subunit B

Goat antiCTB

1:4000

RGC axons

Phosphorylated
heaviest NF
subunit
Brn3a (Pou4f1)

Mouse antiRT97

703. List Biological
Laboratories,
QuadraTech, Surrey, UK
MCA1321. Serotec,
Bionova Scientific,
Madrid, Spain
sc-31984. Santa Cruz
Biotechnologies,
Heidelberg, Germany
7699/4. Swant, Marly,
Switzerland
ab5405. ChemiconMillipore Iberica, Madrid,
Spain
sc-14363. Santa Cruz
Biotechnologies,
Heidelberg, Germany
MCA970. Serotec,
Bionova Scientific,
Madrid, Spain

PA1-780. Pierce,
ThermoFisher, Madrid,
Spain

1:1200

RGCs

Amacrine
cells and
RGCs
L-cones

Goat antiBrn3a (C-20)

Calretinin

Rabbit anticalretinin

Human red/
green opsin

Rabbit antiopsin red/
green
Goat antiOPNS1SW
(N20)
Mouse antirat RECA1
Clone HIS52
Rabbit antimelanopsin
(NH2terminal)

Rabbit antimelanopsin
UF006

S-cones

Blue opsin

Retinal
vessels

Rat endothelial
cell antigen 1

m+RGCs
(rats)

Melanopsin

m+RGCs
(mice)

Melanopsin

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

Working
dilution

1:200

1:750

1:2500

1:1000

1:1000

1:500

1:5000

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 following previously described procedures that are standard in our laboratory (GalindoRomero et al., 2011, 2013a; Nadal-Nicola´s et al., 2009; Salinas-Navarro et al.,
2009a; Valiente-Soriano et al., 2014), using an epifluorescence microscope
(Axioscop 2 Plus; Zeiss Mikroskopie, Jena, Germany) equipped with a computerdriven 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

7


8

CHAPTER 1 Ocular hypertension-induced retinal degeneration

wholemount or cross section was composed of 154 (rat) or 140 (mouse) individual
frames captured side by side with no gap or overlap between them with a 10Â (rat)
or 20 Â (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 automatically, while m+RGCs were counted manually following previously described
methods (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; ValienteSoriano et al., 2014). The topography of all cells, except m+RGCs, was assessed
using isodensity maps (Galindo-Romero et al., 2011; Nadal-Nicola´s et al., 2009;
Ortı´n-Martı´nez et al., 2010, 2014; Salinas-Navarro et al., 2009a). Distribution of
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 inner
retinal vessels, RECA1 signal was transformed into a black (background) and white
(vessels signal) image using the image analysis software IPP (Valiente-Soriano et al.,
2015b). Retinal layer thickness (three sections/retina) was measured in the photomontages with a semiautomated routine developed with IPP macrolanguage
(Ortı´n-Martı´nez et al., 2015). The area occupied with CTB labeling in the contralateral or ipsilateral SC was measured using the image analysis software IPP (ValienteSoriano 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 intense
CTB 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 was
done using SigmaStat® 3.1 for Windows® (SigmaStat® for Windows™ Version
3.11; Systat Software, Inc., Richmond, CA). Kruskal–Wallis was used when comparing more than two groups and Mann–Whitney when comparing two groups only.
Differences were considered significant when p < 0.05.

3 RESULTS AND DISCUSSION
Here, we review some recent studies in which we have addressed several questions
regarding the short- and long-term effects of LP-induced OHT in the adult rodent
retina: (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 population? (iii) Does OHT affect other non-RGC neurons in the GCL? (iv) Does OHT


3 Results and discussion

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 during 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; ValienteSoriano et al., 2015b). In adult albino and pigmented mice, IOP levels raised above
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; ValienteSoriano et al., 2015a). As previously reported, the IOP elevation was somewhat
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 number 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
et al., 2014; Schlamp et al., 2006; Soto et al., 2008). Thus, although these
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,
1981; Salinas-Navarro et al., 2009b). In adult pigmented mice at survival intervals
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 labeling 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


10

CHAPTER 1 Ocular hypertension-induced retinal degeneration

layers of the SC and highest densities of CTB immunoreactivity in the stratum zonale
and stratum griseum superficiale, where RGC axons arborize, differentiate, and establish synaptic contacts with target neurons. In the experimental mice, there was a
marked reduction in the amount of CTB-labeled retinal afferents in the superficial
layers of the contralateral SC; there were small patches in which CTB labeling
was reduced allowing observation of individual axons and their terminal arborizations. In addition, there were areas with little to none CTB immunoreactivity that
often presented the form of a column extending in the dorsoventral axis of the visual

layers of the SC, resembling the deployment of rodent axon terminals (Ling et al.,
1998) and suggesting degeneration of retinal axons and their terminals, as observed
following other types of retinal insults (Avile´s-Trigueros et al., 2003; MayorTorroglosa et al., 2005; Vidal-Sanz et al., 2007). The lateral extension of these areas
varied from a small narrow column to almost one half or more of the SC mediolateral
extension (Fig. 1), whereas the rostrocaudal extension of the SC varied from a few to
almost 10–14 consecutive serial coronal sections. Approximately 50% of the area
occupied by the visual layers of the right SC did not show CTB-labeled retinal terminals (Fig. 1). The amount of this lack of labeling did not progress between 10 days
and 14 weeks, and this is consistent with the observation that RGC loss in this mice
OHT model does not progress during this period of time (Valiente-Soriano et al.,
2015a). Moreover, there was a correlation between the amount of RGC loss in the
retina and the diminution in retinotectal denervation. In adult albino rats, Drouyer
et al. (2008) found a reduction in retinal fiber density in different retinorecipient
structures with a range from approximately 50% in the ventral lateral geniculate nucleus to 72% in the suprachiasmatic nucleus, and 50% in the SC. Our results in adult
pigmented mice are consistent with those found in adult albino rats and further
strengthen the idea that OHT results not only in marked degeneration of the RGC
layer but also in the anterograde degeneration of retinofugal axons and thus in significant denervation of the retinorecipient target nuclei in the brain (Crish et al.,
2010; Dekeyster et al., 2015; Yu¨cel et al., 2003).

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;
Schnebelen et al., 2009; Valiente-Soriano et al., 2015b) as well as in adult albino
(Cuenca et al., 2010; Salinas-Navarro et al., 2009c) and pigmented (ValienteSoriano et al., 2015a) mice, OHT resulted within the first 2 weeks in the loss of approximately 80% of the RGC population identified in the left (lasered) retinas with
the retrograde tracers FG or OHSt applied to both SCi 1 week prior to animal processing. These retinas showed areas that were almost devoid of retrogradely labeled
RGCs and adopted the form of pie-shaped sectors with their base located on the retinal periphery and their apex toward on the optic disc; these areas were more frequent
in the dorsal retinas and varied in size from a small sector to one or several retinal
quadrants. In contrast, the right (control not lasered) retinas showed a normal


3 Results and discussion

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: À3.08 to À4.72 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).

distribution of RGCs (retrogradely labeled or immunostained with Brn3a) with highest 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
et al., 2009a,b). The construction of isodensity maps allowed detailed examination
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 agreement with previous reports from this (Vidal-Sanz et al., 2012) and other (Fu and
Sretavan, 2010; Levkovitch-Verbin et al., 2002) laboratories. Moreover, variability
in the degree of degeneration has also been reported in an inherited pigmented mouse

11


12

CHAPTER 1 Ocular hypertension-induced retinal degeneration

FIGURE 2
Ocular hypertension induces loss of orthotopic and displaced retinal ganglion cells. Maps of
three representative retinas (one per row) showing the distribution of retrogradely traced
orthotopic (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 (second
and 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 and
Brn3a+), which is consistent with an axonal compression produced at the level of the
optic nerve head. At the bottom of each map is shown the number of RGCs or dRGCs
represented. 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) ¼ 1 mm.

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 maps
also revealed a diffuse loss, even within the retinal areas showing surviving RGCs.
This amount of retinal degeneration was based on quantification of RGCs labeled
with retrograde tracers applied to the SCi 1 week prior to animal processing. When
the 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


3 Results and discussion

FIGURE 3 See legend on next page.

13


14

CHAPTER 1 Ocular hypertension-induced retinal degeneration

was a clear mismatch between the numbers of traced RGCs and the numbers of
DTMR+RGCs or Brn3a+RGCs in the same retinas. The numbers of Brn3a+RGCs
were significantly greater than those of traced RGCs at early periods after LP but
not at surviving intervals of 5 weeks or more, indicating that at early time periods
following OHT a large population of surviving RGCs had lost their active retrograde
axonal transport (Agudo-Barriuso et al., 2013a; Vidal-Sanz et al., 2012); such an alteration has been previously observed following other types of retinal or ON injuries
(Lafuente Lo´pez-Herrera et al., 2002; McKerracher et al., 1990). However, between
1 and 5 weeks after LP, the numbers of Brn3a+RGCs diminished significantly, indicating that RGC loss was progressive between 1 and 5 weeks after LP.
It was interesting to observe that a single intraocular injection of 5 mg of BDNF
right after LP resulted in significant greater RGC survival when compared to vehicletreated retinas examined at 12 or 15 days. The numbers of Brn3a+RGCs in the
vehicle-treated retinas examined at 12 or 15 days represented 56% (n ¼ 4) or 45%
(n ¼ 4), respectively, of the control values, whereas for the BDNF-treated retinas
these proportions were 83% (n ¼ 4) or 72% (n ¼ 4) 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
Di Polo, 2012) that have found BDNF, NT4-5, insulin-like growth factor, or glialderived neurotrophic factor to afford transient neuroprotection against injuryinduced 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 ranging
from 1 to 12 weeks after OHT (Salinas-Navarro et al., 2009c, 2010; Vidal-Sanz
et al., 2012) using the RT97 antibody that detects the highly phosphorylated epitope
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 representative
experimental retina 15 days after laser photocauterization of the perilimbar and episcleral
veins, 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 an
experimental 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. Density

color (different gray shades in the print version) scale in A and B bottom right ranges from
0 (purple (black in the print version)) to !3500 RGCs/mm2 or !5000 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 illustrate
that in the retinal sectors with diminished numbers of Brn3a+RGCs there were large
numbers 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) ¼ 1 mm. Scale bar for (C–D) ¼ 50 mm.


3 Results and discussion

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 o0 clock (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.

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 outside 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

15


16

CHAPTER 1 Ocular hypertension-induced retinal degeneration

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 were
RT97+RGCs as well as bundles of RT97+axons. These bundles of RT97+axons were
observed up to 12 weeks after OHT at a time when the vast majority of the RGC
population is already disconnected from their target, suggesting that retrograde degeneration 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
et al., 2008; Vidal-Sanz et al., 2012). Thus, as shown after ON crush (ParrillaReverter et al., 2009b), it is tempting to suggest that it is difficult to predict RGC
survival based on the appearance of the nerve fiber layer of the retina, since the
appearance 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 to
investigate the fate of neurons in the GCL of the retina. In addition to the RGC
population, within the GCL there is a population of displaced amacrine cells as
numerous 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 the
DAPI+nuclei observed in the pie-shaped retinal sectors lacking Brn3a+RGCs are actually displaced amacrine cells (Fig. 3), although a small proportion may correspond
to endothelial cells, astrocytes, and microglia which are known to respond with

proliferation or cell migration (Rojas et al., 2014; Salvador-Silva et al., 2000;
Sobrado-Calvo et al., 2007). To investigate if the retinal vasculature plays a role
in the sectorial RGC loss, the inner retinal vessels were immunolabeled with RECA1
and examined in wholemounts. There were no apparent vascular abnormalities in the
regions that showed FG+ or Brn3a+RGCs nor in regions lacking RGCs that could
account for the sectorial loss of RGCs (Fig. 4; Valiente-Soriano et al., 2015b). If
the 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 following observations: (i) the typical pie-shaped sectors lacking axons and their parent
RGCs, including the small subpopulation of displaced RGCs (Fig. 2); (ii) the presence in those pie-shaped sectors of non-RGC neurons, as observed with DAPI
and calretinin staining (Fig. 3), presumably displaced amacrine cells; and (iii) the
preservation of the normal appearance of the inner retinal vasculature (Fig. 4), are in
agreement with previous studies (Cone et al., 2010; Jakobs et al., 2005; Kielczewski
et al., 2005; Moon et al., 2005) that have indicated selective damage to the RGC
population in the GCL, and overall speak in favor of some type of mechanical
compression-like damage to bundles of axons in the ON head. While the mechanisms
underlying ON injury in glaucoma are not fully understood, among the main lines of
thought are the mechanical and vascular theories. The mechanical theory argues that
the pressure at the level of the ON head would result in direct compression of bundles