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Experimental Eye Research 91 (2010) 273e285
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Experimental Eye Research
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Changes in the inner and outer retinal layers after acute increase
of the intraocular pressure in adult albino Swiss mice
Nicolás Cuenca a,1, Isabel Pinilla b,1, Laura Fernández-Sánchez a, Manuel Salinas-Navarro c,
Luis Alarcón-Martínez c, Marcelino Avilés-Trigueros c, Pedro de la Villa d,
Jaime Miralles de Imperial c, Maria Paz Villegas-Pérez c, Manuel Vidal-Sanz c, *
a
Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, 03690 San Vicente del Raspeig, Alicante E-03080, Spain
Hospital Miguel Servet, Zaragoza, Instituto Aragonés de Ciencias de la Salud, Spain
Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain
d
Departamento de Fisiología, Facultad de Medicina, Universidad de Alcalá, E-28871 Alcalá de Henares, Spain
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 February 2010
Accepted in revised form 25 May 2010
Available online 1 June 2010
In adult albino mice the effects of increased intraocular pressure on the outer retina and its circuitry was
investigated at intervals ranging 3e14 weeks. Ocular hypertension (OHT) was induced by cauterizing the
vessels draining the anterior part of the mice eye, as recently reported (Salinas-Navarro et al., 2009a).
Electroretinographic (ERG) responses were recorded simultaneously from both eyes and compared each
other prior to and at different survival intervals of 2, 8 or 12 weeks after lasering. Animals were processed
at 3, 9 or 14 weeks after lasering, and radial sections were obtained in the cryostat and further processed
for immunocytochemistry using antibodies against recoverin, g-transducin, Protein Kinase C-a (PKC-a),
calbindin or synaptophysin. The synaptic ribbons were identified using an antibody against the protein
bassoon, which labels photoreceptor ribbons and nuclei were identified using TO-PRO. Laser photocoagulation of the perilimbar and episcleral veins of the left eye resulted in an increase in mean intraocular
pressure to approximately over twice its baseline by 24 h that was maintained for approximately five days
reaching basal levels by 1 week. ERG recordings from the different groups of mice showed their a-, b-wave
and scotopic threshold response (STR) amplitudes, when compared to their contralateral fellow eye,
reduced to 62%, 52% and 23% at 12 weeks after lasering.
Three weeks after lasering, immunostaining with recoverin and transducin antibodies could not
document any changes in the outer nuclear layer (ONL) but both ON-rod bipolar and horizontal cells had
lost their dendritic processes in the outer plexiform layer (OPL). Sprouting of horizontal and bipolar cell
processes were observed into the ONL. Fourteen weeks after lasering, protein kinase-C antibodies
showed morphologic changes of ON-rod bipolar cells and calbindin staining showed abnormal horizontal
cells and a loss of their relationship with their presynaptic input. Moreover, at this time, quantitative
studies indicate significant diminutions in the number of photoreceptor synaptic ribbons/100 mm, and in
the thickness of the outer nuclear and plexiform layer, when compared to their fellow eyes. Increased
intraocular pressure in Swiss mice results in permanent alterations of their full field ERG responses and
in changes of the inner and outer retinal circuitries.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
ocular hypertension
adult albino mice
immunohistochemistry
ERG
photoreceptors
bipolar cells
1. Introduction
Previous studies on ocular hypertensive mice models have
focused predominantly on degenerative changes of the retinal
ganglion cell (RGC) population and their axons in the retinal fibre
* Corresponding author. Tel.: þ34 868883961; fax: þ34 868883962.
E-mail address: [email protected] (M. Vidal-Sanz).
1
These authors contributed equally to this work.
0014-4835/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.exer.2010.05.020
layer and the optic nerve (Aihara et al., 2003a,b; Buckingham et al.,
2008; Howell et al., 2007; Jakobs et al., 2005; Salinas-Navarro et al.,
2009a, 2010; Schlamp et al., 2006; Soto et al., 2008). Following the
changes at the RGC population, other retinal neuronal populations
may also be affected (Bayer et al., 2001a,b; Mittag et al., 2000)
including the inner (Panda and Jonas, 1992a) and outer nuclear layers
of the retina (Nork et al., 2000; Panda and Jonas, 1992b). Affectation
of other non-RGC neuronal populations in the retina has been shown
in a number of morphological and functional studies that have used
electroretinogram (ERG) recordings and the measurement of the
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thickness of the inner and outer retinal layers (Fazio et al., 1986; Korth
et al., 1994; Salinas-Navarro et al., 2009a). Experimental models of
elevated IOP (Bui et al., 2005; Chauhan et al., 2002; Feghali et al.,
1991; Fortune et al., 2004; Holcombe et al., 2008; Kong et al., 2009)
have shown important alterations of several ERG components,
including the scotopic threshold response (STR), the a- and b- waves,
which are associated with RGCs, photoreceptors and bipolar cells,
respectively.
To further study the mechanisms by which elevated IOP causes
retinal damage in adult albino mice we have used an ocular
hypertension (OHT) induced retinal damage model, based on laser
photocoagulation of the perilimbal and episcleral veins (SalinasNavarro et al., 2009a, 2010). A recent study has shown in adult
albino Swiss mice that lasering of the limbal tissues results in rapid
elevation of the IOP which in turn results shortly after in diffuse as
well as focal loss of RGCs, that adopted the form of pie-shaped
wedges with their vertex located towards the optic nerve, and
exhibited variable sizes ranging from a small sector to several
quadrants of the retina (Salinas-Navarro et al., 2009a). Moreover,
following elevated IOP there were permanent diminutions of the
scotopic threshold response (STR) associated with the loss of the
great majority of RGCs, indicating the value of noninvasive functional ERG losses associated with RGC loss in adult rodents (AlarcónMartínez et al., 2009; Salinas-Navarro et al., 2009a). In addition there
were also permanent diminutions of the major components of the
ERG, the a- and b- waves, as well as lengthening of their implicit
times, all of which imply severe alterations of the inner and outer
nuclear layers of the retina (Salinas-Navarro et al., 2009a). In the
present studies we have extended previous observations and have
further studied short and long-term, qualitatively and quantitatively,
functionally and morphologically, the retinal changes secondary to
OHT in adult albino Swiss mice, not only at the outer retinal neurons
but also at the neurotransmitter level. We present evidence indicating that following laser injury to the aqueous outflow pathways in
the Swiss mice there is a functional impairment as well as retinal
degeneration that affects the inner and outer synapse and nuclear
layers, resulting in alterations of the inner and outer retinal circuitries. Short accounts of this work have been published in abstract
form (Pinilla et al., 2008; IOVS ARVO-E-Abstract 5482).
2. Methods
2.1. Animals: handle and care
Experiments were performed on 15 adult albino Swiss male
mice (40e45 g), obtained from the breeding colony of the University of Murcia (Murcia, Spain). Mice were housed in temperature
and light controlled rooms with a 12 h light/dark cycle and had food
and water ad libitum. Light intensity within the cages ranged from 9
to 24 luxes. Animal manipulations followed institutional guidelines, European Union regulations for the use of animals in research
and the ARVO statement for the use of animals in ophthalmic
and vision research. All surgical manipulations were carried out, as
previously described (Salinas-Navarro et al., 2009b,c), under
general anesthesia induced with an intraperitoneal (i.p.) injection
of a mixture of ketamine (75 mg/kg, KetolarÒ, Parke-Davies, S.L.,
Barcelona, Spain) and xylazine (10 mg/kg, RompúnÒ, Bayer, S.A.,
Barcelona, Spain). At the end of the lasering treatment, retinal
blood flow was assessed by examination of the eye fundus through
the operating microscope (Vidal-Sanz et al., 2001, 2007; AvilésTrigueros et al., 2003). During recovery from anaesthesia, mice
were placed in their cages, and an ointment containing tobramycin
(TobrexÒ, Alcon S.A., Barcelona, Spain) was applied on the cornea
to prevent corneal desiccation. Additional measures were taken
to minimize discomfort and pain after surgery. Animals were
sacrificed with an i.p. injection of an overdose of pentobarbital
(Dolethal VetoquinolÒ, Especialidades Veterinarias, S.A., Alcobendas, Madrid, Spain).
2.2. Induction of ocular hypertension
Ocular hypertension (OHT) was induced by cauterizing the
vessels draining the anterior part of the mice eye, as recently
reported (Salinas-Navarro et al., 2009a). In brief, on anesthetized
mice the left eyes were treated on a single session with a combination of diode laser (532 nm, Quantel Medical, Clermont-Ferrand,
France) burns. Laser beam was directly delivered without any lenses,
aimed to the limbal and epiescleral veins. Special care was taken to
avoid the damage to the cilliary body and other blood vessels.
The spot size, duration and power were 50e100 mm, 0.5 s and 0.3 W
respectively. Mice received each approximately 72 spots, in a single
session. Animals were housed in temperature and light controlled
rooms with a 12 h light/dark cycle and had food and water ad libitum.
2.3. IOP measurement and experimental groups
The IOP was measured under anesthesia in both eyes with
a rebound tonometer (Tono-LabÒ, Tiolat, OY, Helsinki, Finland)
(Danias et al., 2003; Salinas-Navarro et al., 2009a) prior to surgery,
and at different time intervals after laser treatment. At each time
point, 36 consecutive readings were carried out for each eye,
averaged and shown as means SD for each time examined. To
avoid fluctuations of the IOP due to the circadian rhythm in albino
Swiss mice (Aihara et al., 2003b) or to the elevation of the IOP itself
(Drouyer et al., 2008), IOP was always tested around the same time,
in the morning and right after deep anesthesia. Moreover, because
general anesthesia lowers IOP in rodents we measured the IOP
treated eye as well as the contralateral intact fellow eye in all the
experiments. Animals were divided into several groups sacrificed at
increasing survival intervals of 3 (group I, n ¼ 5), 9 (group II, n ¼ 4)
or 14 (group III, n ¼ 6) weeks after lasering.
2.4. ERG recordings
Prior to ERG recordings, animals were dark adapted overnight
and their manipulation was done under dim red light (l > 600 nm),
as recently described (Salinas-Navarro et al., 2009a). In brief, mice
were anaesthetized, their IOP was measured as above, and bilateral
pupil midriasis was induced by applying in both eyes a topical drop
of 1% tropicamide (Tropicamida 1%Ò, Alcon-Cusí, S.A., El Masnou,
Barcelona, Spain). The light stimulation device consisted in Ganzfeld
dome, which ensures a homogeneous illumination anywhere in the
retina, with multiple reflections of the light generated by light
emitting diodes (LED), which provided a wide range of light intensities. For high intensity illuminations, a single LED placed close
(1 mm) to the eye was used. The recording system was composed by
BurianeAllen bipolar electrodes (Hansen Labs, Coralville, IA, USA)
with a corneal contact shape; a drop of methylcellulose 2%
(Methocel 2%Ò; Novartis Laboratories CIBA Vision, Annonay, France)
was placed between the eye and the electrode to maximize
conductivity of the generated response. The reference electrode was
placed in the mouth and the ground electrode in the tail. Electrical
signals generated in the retina were amplified (1000) and filtered
(band pass from 1 Hz to 1000 Hz) by the use of commercial amplifier
(Digitimer Ltd, Letchworth Garden City, UK). The recorded signals
were digitized (Power Lab; AD Instruments Pty. Ltd., Chalgrove, UK)
and displayed on a PC computer. Bilateral ERG recording were
performed simultaneously from both eyes.
Light stimuli were calibrated as follows before each experiment
and the calibration protocol assured the same recordings parameters
N. Cuenca et al. / Experimental Eye Research 91 (2010) 273e285
for both eyes. The stimuli device was formed by 3 independent
circuits of 4, 8 or 16 white LEDs, placed over the Ganzfeld dome. The
illumination was controlled by a computer which sent an electrical
current to one of the LED circuits. Before the actual experiment,
the stimuli device was calibrated by a photometer situated inside
the Ganzfeld dome, in approximately the same position were the
animals head was placed. We presented a series of stimuli (currents)
for each LED-circuit, along a wide intensity range, and we obtained
the corresponding photometer readings (cd/m2) for each stimulus.
According to the time exposed to the stimulus presented to the
animal, we multipled the value of the light (cd/m2) by the stimulus’
exposure time (s) and we obtained the logarithm of the result (log
unit cds/m2).
The scotopic ERG responses were recorded by stimulating the
retina with light intensities ranged between 105 and 104 cd s m2
for the scotopic threshold response (STR), 104 and 102 cd s m2 for
the rod mediated response and between 102 and 100.5 cd s m2 for
the mixed (response mediated for rods and cones) response. For
each light intensity, a series of ERG responses were averaged and the
interval between light flashes was adjusted to appropriate times that
allowed response recovering. At the end of each session the animals
were treated with topical tobramicine (TobrexÒ, Alcon-Cusí, S.A., El
Masnou, Barcelona, Spain) in both eyes. The analysis of the different
recordings was performed with the normalization criterions established for the ISCEV for the measures of the amplitude and implicit
time of the different waves which were studied (Salinas-Navarro
et al., 2009a). ERG were recorded prior to lasering and at 2, 8 or 12
weeks after lasering for groups I (n ¼ 5), II (n ¼ 4) or III (n ¼ 6),
respectively.
The a-wave was measured from the baseline to the first valley, ca.
10 ms, from the flash onset and the b-wave amplitude was measured
from the bottom of the a-wave trough to the top of the hill of the
positive deflection; the time point of the b-wave measurement,
varied depending of the intensity used. The implicit time was
measured from the presentation of the stimulus to the peak of the bwave. Data from operated and non-operated eyes were compared;
ERG wave amplitudes and implicit times were calculated for each
animal group and the percentage of difference between the operated
and the non-operated eyes were obtained for each stimulus and
further averaged (mean SEM). The results were analyzed with
SigmaStatÒ 3.1 for WindowsÒ (Systat Software, Inc., Richmond, CA,
USA). Descriptive statistics were calculated and t-test was used in all
studied groups for the comparison between prior and post lasering.
We performed the t-test prior to surgery to compare the response of
both eyes to demonstrate similar functionality in both eyes prior to
the lesion. The statistic significance was placed in a p < 0.05 for all
tests and the statistic was always of two tails.
2.5. Immunocytochemical studies
Animals were studied at 3, 9 and 14 weeks after lasering.
Animals were sacrificed and eyes were enucleated. The eyecups
were fixed in 4% paraformaldehyde in 0.1 M PBS at pH 7.4 for 1 h
and then washed in 0.1 M PBS before being cryoprotected in 15%
sucrose for 1 h, 20% sucrose for 90 min and 30% sucrose overnight
at 4 C. Next day they were embedded in OCT and cross sections of
the retina were cut at 16 mm thickness on a cryostat in a horizontal
plane, and mounted on glass slides. Sections were treated as in
previous studies (Cuenca et al., 2002, 2004, 2005a,b) for immunostaining, using as primary antibodies mouse anti-recoverin
1:1000 (Dr. McGinnis), rabbit anti-g-transducin 1:500 (Cytosignal),
rabbit anti-PKC-a 1:100 (Santa Cruz), rabbit anti-Calbindin (Swant),
mouse anti-synaptophysin 1:300 (Sigma). The synaptic ribbons
were identified using an antibody against the protein bassoon,
which labels photoreceptor ribbons (mouse anti-bassoon 1:5000;
275
Stressgen). Nuclei were identified using TO-PRO 1:1000 (Molecular
Probes). Slides were mounted in watermount (Vector Labs) and
coverslipped for viewing by confocal microscopy (Leyca TCS SP2).
To control for non-specific staining, some sections were stained
omitting the primary antibody.
Qualitative examination of the sections revealed differences
between injured and control retinas that were more marked in the
group of animals examined 14 weeks after laser, thus for this group
we have quantified in a masked fashion: i) the thickness of the outer
nuclear layer (ONL) and outer plexiforme layer (OPL) using a TO-PRO
stain which labels nucleus of retinal cells, and ii) the number
of synaptic contacts between photoreceptors and bipolar cells, by
counting the number of spots bassoon immunopositive. Bassoon
labels the synaptic ribbon at the axon terminal of photoreceptors,
and each bassoon spot in the OPL is associated with its corresponding bipolar dendrite tip pairing. Cross sections from the central
retina, close to the optic nerve head, in 5 animals, were photographed. At least seven pictures from different alternate sections at
63 of magnification for TO-PRO images, and 100 of magnification
for bassoon immunostained retinas, were obtained from each
experimental and its corresponding right (non-lasered) fellow eye,
which was used as a control. The number of spots bassoon positive
was expressed as a mean of labeled cells per 100 mm. The thickness
of the ONL and OPL were measured using ImageJ NIH program and at
least thirty linear measures were done for each picture. For statistical
analysis, the Student t-test was performed with Prism 5.01 program
(GraphPad Software, La Jolla, CA. USA).
3. Results
3.1. Laser-induced IOP values
There was some variability among the maximum IOP values
registered from the lasered-eyes, within each of the animals and
the subgroups processed at different survival intervals, but in
general the results were rather consistent (Fig. 1AeC).
Overall our data show a similar time course elevation of the IOP
for all groups of mice analyzed (Fig. 1AeC). There was an important
increase of the IOP by 24 h after lasering, to twice its baseline value
that remained elevated for the following four days and returned to
baseline values by one week after lasering. Indeed, the statistical
analysis showed that the IOP values were comparable among the
different groups and eyes (KurskaleWallis test, p ¼ 0.3547). Twenty
four hours after lasering, the IOP values were comparable within
the different animal groups in the lasered (KruskaleWallis test,
p ¼ 0.2546) as well as in the contralateral uninjured right eyes
(KruskaleWallis test, p ¼ 0.5827). There was a substantial increase of
the IOP that was already evident 24 h after lasering (ManneWhitney
test, p ¼ 0,0011) and was maintained up to the fifth day (ManneWhitney test, p ¼ 0,0011), returning to their basal values by one
week after lasering when the IOP values in animals of the different
groups were comparable for both eyes (KruskaleWallis test,
p ¼ 0.1859) (Fig. 1C).
3.2. ERG responses
3.2.1. ERGs in control albino mice
To study the effect of elevation of the IOP on the ERG waves in
albino mice, simultaneous ERG recordings were performed from
right and left eyes of each animal prior to surgery. No significant
differences were observed in the a- or b-wave ERG amplitudes,
between the left and right eyes in any of the animals of this study
prior to surgery (Fig. 2A). Representative examples of the ERG
traces recorded in both eyes, prior to surgery, in response to flash
stimuli of increasing intensity are shown in Fig. 3. No ERG a-wave
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N. Cuenca et al. / Experimental Eye Research 91 (2010) 273e285
Fig. 1. Intraocular pressure measurements in adult albino mice. AeC Histogram shows
mean (SD) intraocular pressure (IOP) values in for the left (lasered) eye and right
(control) eye in groups I (A), II (B) and III (C) of mice that were sacrificed 3, 9 or 14
weeks, respectively, after laser photocoagulation of the limbal and episcleral veins of
the left eye. Each received an average of 72 laser-burning spots in a single session. IOP
values in group I (A) rose by day 1 post-treatment to twice their normal values and
returned to basal preoperative levels by 1 week. IOP values in group II (B) followed
a similar pattern of pressure variations. IOP values in group III (C) also showed by 1d
a raise to twice their normal values, that were maintained for the following four days
and decreased gradually to basal levels by one week, remaining at basal levels for the
rest of the study. Thus, IOP elevation in all these three groups was not maintained
beyond 1 week.
was observed for light intensities below 2 log cds/m2. The b-wave
elicited by light intensities from 3.66 to 0.5 log cds/m2 increased
exponentially (Fig. 3).
3.2.2. ERGs in experimental albino mice after elevated IOP
ERG recordings were performed simultaneously from right nontreated and left lasered eyes in albino mice at specific survival
intervals after lasering. A representative example from group I mice
registered 2 weeks after lasering shows the STR and scotopic a- and
Fig. 2. Electroretinographic amplitude measurements in albino mice. Bar histogram
showing averaged data (mean SEM) for the right and left eye a-wave and b-wave
ERG amplitudes versus stimulus intensities from the groups of mice studied. A shows
a- and b-wave ERG amplitudes prior to laser from a representative group of animals.
No significant differences were observed between both eyes (T-test, p > 0.05). B, C and
D show a- and b-wave ERG amplitudes at 2, 8 or 12 weeks after lasering respectively.
N. Cuenca et al. / Experimental Eye Research 91 (2010) 273e285
277
Fig. 4. Changes in ERG measurements at different survival intervals. Histogram plots
illustrating changes in the amplitudes of the ERG components studied (a-, b-waves and
pSTR), recorded from the experimental groups analyzed 2, 8 or 12 weeks after lasering.
Measurements for all showed light intensity stimulus were averaged (mean SEM) for
the a-, b-waves and pSTR, and presented as % of their fellow eyes. For the groups
studied at 2, 8 or 12 weeks after laser treatment, the a-, b-wave and pSTR amplitude
were significantly reduced * (T-test; P < 0.05) to approximately 73, 52 and 44% or 73,
72 and 50% or 62, 52 and 23% with respect to their fellow eyes.
Fig. 3. Scotopic electroretinographic recordings in albino mice. Examples of the ERG
traces recorded in an albino mice in response to flash stimuli of increasing intensity
(indicated in log cds/m2 units at the left of the recording traces) for the right eye
(thin trace) and for the left experimental eye (bold trace). STR were elicited by light
intensities of 4.26 log cds/m2 and rod and mixed responses were elicited by light
intensities from 3.66 to 0.59 log cds/m2. Vertical arrows denote presentation of
light stimulus. No significant difference in the ERG amplitudes between left and right
eyes were observed prior to laser (T-test, p > 0.05). However, at 2 week after lasering
the limbal tissues, there is a clear reduction in all studied waves from the left eye
(T-test, p < 0.001).
b-waves siginificantly reduced when compared to the pre-lasering
recordings (Fig. 3). In addition, data are shown as averages of
absolute wave amplitudes (mean SEM) in lasered and contralateral fellow eyes (Fig. 2BeD). ERG recordings from the different
groups of mice showed average reductions in the scotopic and
mixed ERG recorded at all survival intervals examined. Their a-,
b-wave and pSTR amplitudes, when compared to their contralateral
fellow eye, were reduced to approximately 73%, 52% and 44%, 73%,
72% and 50%, or 62%, 52 and 23% of their control values at 2, 8 or 12
weeks after lasering, respectively (Fig. 4). These values were
significantly smaller than control values obtained prior to lasering
(t-test, p < 0.05 for all times and waves).
Overall, these figures illustrate significative reductions in the a-,
b-wave and pSTR amplitudes that were obvious already by 2 weeks
after lasering, and persisted for up to 12 weeks, the longest time
interval recorded in the present studies, indicating that these
functional alterations were permanent.
3.3. Immunofluorescence findings
3.3.1. Three weeks after acute IOP increase
Photoreceptors were identified using recoverin (McGinnis et al.,
1999) and their nuclei were stained using TO-PRO. Three weeks
after lasering no remarkable changes were found at the photoreceptor level. Photoreceptor nuclei labeled with TO-PRO maintained
a similar number of rows, although some of the cell bodies located at
the inner part of the outer nuclear layer (ONL) had lost their alignment and were lightly misplaced into the outer plexiform layer (OPL)
and into the photoreceptor segments layer (Fig. 5A and B arrows).
Using antibodies against g-transducin that specifically label cones
we observed that transducin immunoreactivity in normal mice
was particularly prominent in the cone outer segments, cone inner
segments and cell bodies, while the pedicles were more lightly
stained. There were some subtle differences in cone morphology
between the lasered and control eyes (Fig. 5C and D). In the untreated
mouse eyes, cone cell bodies stained with transducin were confined
to the outer third of the ONL, whereas in the lasered eyes some of the
cone cell bodies were displaced towards the middle portion of the
ONL (Fig. 5D, arrows). In most studied retinas no differences were
found in the cone outer segment length and morphology. However,
the shape of cone pedicles in the lasered eyes looks somewhat flattened when compared to control retinas (Fig. 5C, D).
To study if the IOP increase induces retinal changes at the OPL
level, we stained ON rod bipolar cells using antibodies against PKC-a.
At this time point, bipolar cell dendrites showed the first signs of
OHT-induced retinal damage; there was a loss of the apical dendrites
and their dendritic terminals appeared to be reduced in length and
in density (Fig. 5F, arrows), although their cell body morphology,
axon and axon terminal appeared normal (Fig. 5E and F).
Other cells that establish synaptic contacts with the photoreceptors at the OPL level are the horizontal cells. Horizontal cells can
be identified using antibodies against calbindin. In normal mouse
retinae, the dendrites of the single horizontal cell type, the B-type
cell, contact cone terminals, and the axon terminal contacts rod
terminals. In the lasered eyes, changes were also remarkable at the
horizontal cells. The regular and dense plexus of horizontal cells
processes in the OPL was lost and there was a clear reduction of
dendrites and axon terminal tips (Fig. 5G and H). In the retinas of
the treated eyes, horizontal cell processes showed clear sprouting
and some of their terminal ends were found inside the ONL (Fig. 5H
arrows).
3.3.2. Nine weeks after lasering
Nine weeks after lasering the number of photoreceptor rows, as
seen with TO-PRO and recoverin, were diminished. The decrease
was obvious comparing the ONL thickness of the normal mice and
the lasered mice at this time point (control versus the lasered eye)
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Fig. 5. Retinal changes three weeks after acute IOP increase. (A and B) Cross section of retinas labeled with TO-PRO. Cross-retina sections showed no remarkable differences in
retinal layer thickness between control (A) and laser treated eyes (B). (C and D) Vertical sections of retinas stained with antibodies against g-transducin showed some subtle cone
morphological differences between untreated (C) and treated eyes (D). Some displaced cone cell bodies in the center of the ONL could be observed (B, arrows). (E and F) PKCa immunostaining to study ON rod bipolar cells. Loss of dendritic terminal (arrows) could be observed in treated retinas (F) compared with non treated eyes (E). (G and H)
Horizontal cells immunostained with antibodies against calbindin. In treated eyes, the retina showed a decrease of horizontal cells processes and a loss of their terminal tips
(arrows) (H).
(Fig. 6A and E versus 6B and F). In the untreated eyes of the normal
mice the staining with recoverin and TO-PRO showed 12- to 13photoreceptor rows forming the ONL while in the laser-treated eye
there was an average of 8e9 photoreceptor rows. Rod bipolar cell
dendrites immunostained with antibodies against PKC-a were
diminished and showed less complex branching patterns in treated
eyes (Fig. 6D) compared with control ones (Fig. 6C). In the retinas of
the lasered eyes, some sprouting of rod bipolar cells dendrites could
be observed at the ONL level (Fig. 6D, arrows). In addition to
photoreceptor staining, recoverin antibodies immunostained two
types of cone bipolar cells the ON-cone bipolar Type 8 and OFFcone bipolar Type 2; both of them showed a decrease of immunoreactivity in treated eyes (Fig. 6F) compared to untreated eyes
(Fig. 6E).
N. Cuenca et al. / Experimental Eye Research 91 (2010) 273e285
279
Fig. 6. Retinal cell changes nine weeks after lasering. (A and B) Cross section of retinas labeled with TO-PRO showing in the treated eyes a decrease of photoreceptors rows in the
ONL. Control retinas had an average number of 12e13 rows (A) compared with 8e9 rows in treated eyes (B). (C and D) Rod bipolar cell immunostained with antibodies against PKCa. Rod bipolar cell dendrites were fewer in number and showed less complex branching patterns in treated eyes (D) compared with control retina (C). In lasered retinas some
sprouting of bipolar cells dendrites into the ONL could be observed (D, arrows). (E and F) Vertical sections showing horizontal cells labelled with calbindin antibody (green) and
photoreceptor and cone bipolar cells labelled with antibody against recoverin (red). There was a loss of horizontal cell processes and their contacts with photoreceptors terminals in
treated retinas (F). At this time sprouting of horizontal cells processes into the ONL was evident (F, arrow). In treated eyes (F) a loss of recoverin immunoreactivity in cone bipolar
cells at the INL was observed compared with control retina (E). Scale bar represents A, B, E, F ¼ 20 mm, C, D ¼ 10 mm.
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N. Cuenca et al. / Experimental Eye Research 91 (2010) 273e285
Nine weeks after lasering, horizontal cell dendrites showed
a decrease of processes in the OPL and some sprouting into the ONL
(Fig. 6F, arrow in inset). At this time no major changes were found
at the inner retina, the inner plexiform layer (IPL) thickness
remained normal and the three plexus stained with antibodies
against calbindin could be identified.
3.3.3. Fourteen weeks after lasering
A clear reduction of neurons in the ganglion cell layer were found
in treated eyes stained with TO-PRO (Fig. 7C and D) compared with
non treated eyes (Fig. 7A and B). The same reduction of neurons in
the ganglion cell layer was observed when the retinas were stained
with antibodies against calbindin (Fig. 7A and C, arrows).
Fourteen weeks after lasering TO-PRO staining showed a clear
diminution in the number of photoreceptors (Fig. 7A and C).
The thickness of photoreceptor layer was markedly reduced in size
when compared to the untreated eye (compare Fig. 7D versus 7B
and see below).
In laser-treated mouse retinas, ON rod bipolar cells dendritic
branches (Fig. 8B) were less profuse and some cells were devoid of
dendrites compared with control retinas (Fig. 8A). An obvious
decrease of cell number, immunoreactivity and dendritic terminals
were found in treated eyes (Fig. 8B). A double immunolabelling with
the antibody against the presynaptic protein bassoon which labels
photoreceptor synaptic ribbon terminals in rod spherules and cone
pedicles in the OPL allowed examining their synaptic contact distribution with ON rod bipolar cells immunostained with antibodies
against PKC-a (Fig. 8A and B). In the retina of the control eyes, ON rod
bipolar cell dendrite tips (green) were all associated with horse
shoes-like bassoon immunoreactivity (red) (Fig. 8A). In the lasered
eyes 14 weeks after the treatment, the more remarkable change was
the loss of bipolar cell dendrites and a diminution in the number of
bassoon immunoreactive spots; both findings suggest a loss of
contact between ON rod bipolar cell dendrites and the photoreceptor
synaptic axon terminals. Some bassoon immunoreactive spots were
found at the ONL layer with no bipolar cell contacts (Fig. 8B).
This reduction was associated with a decrease of the OPL
thickness (Figs. 8A, B and 9A). In order to assess if this change was
due to the loss of the photoreceptor axon terminals we carried out
a double staining for calbindin and synaptophysin (SYP), a synapticvesicle protein present in the photoreceptor axon terminal that was
used to identify the presynaptic elements of pedicles and spherules
of their respective photoreceptor types. A close relationship
was found at the OPL between SYP photoreceptor axon endings and
horizontal cells terminals (Fig. 8C). In normal retinae, the dendritic
tips of the horizontal cells (labeled with calbindin) were associated
with rod spherules (labeled with SYP). SYP labeling was continuously distributed across the OPL (Fig. 8C). In the retina of the treated
eyes, there was a dramatic reduction of SYN immunoreactivity
(Fig. 8D) showing a great loss of photoreceptor axon terminals that
Fig. 7. Low magnification cross section retinas 14 weeks after lasering, double labeled with TO-PRO and antibodies against calbindin (green). A reduction of photoreceptor rows
were found comparing control (A,B) and treated eyes (C,D). Significant ganglion cells loss labeled with TO-PRO were also observed comparing control B and treated eyes D.
Immunopositive calbindin ganglion cells were decreased in treated eyes (C arrows) versus control retina (A arrows). ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion
cell layer. Scale bar ¼ A and C 200 mm, B and D ¼ 20 mm.
N. Cuenca et al. / Experimental Eye Research 91 (2010) 273e285
281
Fig. 8. Confocal fluorescence micrographs of retinal cross sections showing synaptic contacts between bipolar and horizontal cells with photoreceptors in the OPL 14 weeks after lasering
(B,D,F) compared with control retinas (A,C,E). (A and B) Confocal fluorescence micrographs of a retinal cross section showing a loss of photoreceptor ribbon synapses with ON rod bipolar
cell at the OPL level in the retina of treated eyes (B). The synaptic ribbons were visualized with an antibody against the protein bassoon (red) which labels photoreceptor ribbons in both
cone pedicles and rod spherules. Bipolar dendrites were labeled with an antibody against PKC-a. In treated eyes bipolar cell (B) dendritic branches were less profuse and some cells were
devoid of their dendrites. A few contacts between bipolar terminals and photoreceptor axon terminals immunostained with bassoon (red) were observed. (C and D) Horizontal cell
dendrites stained with antibodies against calbindin and photoreceptor axon terminal immunostaining with antibodies against synaptophysin. In control animal, using SYN staining, 3 to 4
axon terminal rows could be recognized (C). In treated eyes a diminution of the axon terminals could be observed (D, arrows). (E and F) In retinas of the treated eyes at the OPL level and
using calbindin antibodies a loss of the horizontal cell dendrites and axon terminals could be observed (F, arrows) compared with control retina (E). Scale bar represents A-B 20 mm, C-F
10 mm.
were missing their contacts with the few remained horizontal cells
terminal tips (Fig. 8D, F).
Using a TO-PRO stain the mean thickness of the outer nuclear
layer (ONL) was 37.24 1.74 mm (mean SEM; n ¼ 5) for the OHT
eyes and 45.88 6.10 mm (n ¼ 5) for the fellow eyes (Fig. 9A).
Similarly the mean thickness of the outer plexiform layer (OPL) was
7.52 1.48 mm (n ¼ 5) for the OHT eyes and 10.58 0.81 mm (n ¼ 5)
for the fellow eyes (Fig. 9B). There were significant differences for
both measurements (Unpaired t test, p ¼ 0.0159 and p ¼ 0.0038 for
ONL and OPL, respectively).
The number of synaptic contacts between photoreceptors and
bipolar cells were compared in treated and fellow eyes by counting
the number of bassoon immunoreactive spots, expressed as a mean
of labeled cells per 100 mm. This measurement was significantly
greater (Unpaired t test, p ¼ 0.0060) in the control 82.65 10.61 mm
(mean SEM; n ¼ 5) than in the OHT eyes 62.76 5.59 mm (n ¼ 5)
(Fig. 9C).
4. Discussion
Various types of retinal injury have been proposed to study adult
rodent RGCs in an effort to correlate their findings with the human
glaucomatous optic neuropathy. These include optic nerve axotomy
(Aguayo et al., 1987; Chidlow et al., 2005; Nadal-Nicolás et al., 2009;
Parrilla-Reverter et al., 2009a,b; Whiteley et al., 1998), transient
ischemia of the retina (Avilés-Trigueros et al., 2003; Lafuente et al.,
2002; Lopez-Herrera et al., 2002; Mayor-Torroglosa et al., 2005;
Vidal-Sanz et al., 2001, 2007), or elevated IOP (Salinas-Navarro
et al., 2009a, 2010). Because, one of the most important risk
factors associated with human glaucoma is elevated IOP (Kass et al.,
2002; Nouri-Mahdavi et al., 2004; The AGIS Investigators, 2000),
animal models of ocular hypertension (OHT) have attracted much
attention from investigators in an effort to correlate their findings
with human glaucoma, and these models have proven useful to
progress our understanding of OHT-induced RGC and optic nerve
282
N. Cuenca et al. / Experimental Eye Research 91 (2010) 273e285
Fig. 9. Histograms at 14 weeks after lasering showing the differences in width of the ONL (A) and OPL (B) in the laser-induced ocular hypertensive eyes compared with their fellow
eyes, measured in retinal vertical sections stained with TO-PRO. The differences in number of synaptic ribbon identified by bassoon immunoreactive spots in the OPL between lasertreated eyes and control are shown in (C). Untreated eye (right eyes) is represented by black bars and experimental (left eyes) are presented in grey bars. Bars indicate means SEM.
*p 0.05 **p 0.01. ONL outer nuclear layer. ONL outer plexiform layer.
pathology that follows ocular hypertension in inherited
(Buckingham et al., 2008; Filippopoulos et al., 2006; Howell et al.,
2007; Jakobs et al., 2005; Schlamp et al., 2006; Soto et al., 2008)
as well as in experimentally induced ocular hypertension in rats
(Levkovitch-Verbin et al., 2002; Salinas-Navarro et al., 2010;
WoldeMussie et al., 2001) and mice (Aihara et al., 2003a; Fu and
Sretavan, 2010; Grozdanic et al., 2003b; Holcombe et al., 2008; Ji
et al., 2005; Mabuchi et al., 2004; Salinas-Navarro et al., 2009a).
However, much less information is available regarding the functional, structural and molecular changes secondary to OHT, and in
particular this information is lacking in the adult albino Swiss mice.
In the present studies, using electrophysiological and immunofluorescence techniques we have further examined from 3 to 14
weeks, the effects of ocular hypertension (OHT) on the function and
structure of the adult albino Swiss mice retina. Our functional
results show that there are major reductions in the amplitudes of a-,
b-wave and pSTR, which persist for as long as 12 weeks, indicating
a permanent damage to the outer and inner nuclear layers.
Morphologically, we observed changes at the level of the OPL that
appear as early as 3 weeks, progressed with time and by 14 weeks
there was loss of photoreceptors, displacement of other cell types as
horizontal cells, and a diminished relationship between photoreceptors and horizontal cells. Moreover, at this time quantitative
analysis of our morphological data (Fig. 9) indicate significant
diminutions in the number of photoreceptor synaptic ribbons and
in the thickness of the ONL and OPL. Altogether the morphological
data could justify the permanent functional impairment observed
in the ERG recordings.
In agreement with our recent report (Salinas-Navarro et al.,
2009a) the IOP raised up to approximately over twice their basal
values within 24 h, remained elevated for the following 4 days and at
one week the IOP values returned to basal levels. Variability of the
IOP is a common finding in rodent models of glaucoma (Morrison
et al., 1997, 2005, 2008) because different lasering methods result
in different IOP elevation profiles with variations in the peak IOP
value, its latency and total duration of the OHT (Chauhan et al., 2002;
Danias et al., 2006; Levkovitch-Verbin et al., 2002). Similar abrupt
elevations of the IOP were observed for pigmented mice in which the
trabecular meshwork and the episcleral veins were photocoagulated
with laser (Grozdanic et al., 2003a, 2004). However the pigmented
mice of Grozdanic and colleages (2004) showed persistent IOP
increases for over 30 days, while in our study the IOP elevation was
restricted to the first week. Thus, our present study may be regarded
as an acute or subacute OHT model, similar to a recent report in
albino mice (Fu and Sretavan, 2010), and this may be seen as
a disadvantage when compared to a more chronic model of IOP
elevation. Nevertheless our IOP profile produced a severe injury to
the retina that resulted in a number of features, such as sectorial loss
of RGCs and degeneration of the nerve fiber layer (Salinas-Navarro
et al., 2009a) that are typically found in an inherited mice model
of ocular hypertension (Buckingham et al., 2008; Jakobs et al., 2005;
Schlamp et al., 2006; Soto et al., 2008) thus arguing in favour of
a similar damaging mechanism. Moreover, our present studies
highlight the fact that only long-term studies may show the full
consequences of ocular hypertension, even when this was induced
for a short period of time, as in the present studies. Indeed, many of
the previous reports are short-term OHT studies, and this might
explain why they did not find evidence of photoreceptor or any other
non-RGC retinal damage.
In this study we have focused on the OHT induced changes in non
retinal ganglion cell neurons, and thus we have studied functionally
and morphologically the synaptic and nuclear inner and outer layers
of the retina. In our present experiments analyzed at 2, 8 or 12
weeks, we found that shortly after elevation of the IOP, by 2 weeks,
there was an alteration in the amplitudes of the a-, b-waves and pSTR
of the ERG, and these results are analogous to those recently
reported in a parallel study (Salinas-Navarro et al., 2009a) in which
ERG were recorded up to 8 weeks. Our present results are also in
agreement with those registered up to 30e40 days in OHT studies in
pigmented mice (Grozdanic et al., 2003a,b, 2004; Holcombe et al.,
2008) and with those registered 5 weeks after acute elevation of
the IOP (Fortune et al., 2004). Moreover, our findings correlate well
with the overall anatomical and structural changes found in a DBA/
2NNia mice study showing a progressive thinning of the outer retinal
layers and corresponding decreases of the a- and b-wave amplitudes
in the ERG (Bayer et al., 2001a,b; Mittag et al., 2000). Other studies
however, have reported reversible changes in a- and b- wave
amplitudes when IOP values returned to basal levels (He et al., 2006).
More recent studies, using acute IOP increases indicate a threshold
for retinal damage resulting in permanent alterations of the a- and bwaves after a substantial increase of the IOP (above 30e70 mm Hg)
or when the cumulative time-IOP elevation integral is severe (Bui
et al., 2005; Fortune et al., 2004; He et al., 2006; Kong et al., 2009).
In our study the IOP normalized within a week after lasering, but the
N. Cuenca et al. / Experimental Eye Research 91 (2010) 273e285
ERG waves persisted with diminished values for as long as 12 weeks,
thus it is possible that in our experiments, the IOP profile resulted in
severe retinal damage that affected not only the RGC population and
their axons as recently reported (Salinas-Navarro et al., 2009a, 2010),
but also the inner and outer retinal layers, which are responsible for
the genesis of the a- and b-waves. Indeed, the ERG b-wave has been
widely used to diagnose various types of retinal degeneration
related to photoreceptor and/or inner retinal cells in animal studies
(Peachey and Ball, 2003) and its amplitude correlates directly
with ON-bipolar activity (Stockton and Slaughter, 1989; Tian and
Slaughter, 1995) and as such is most likely, in cases of photoreceptor dysfunction, to be directly related to the level of transmission
from photoreceptors to ON bipolar cells via mGluR6 receptors.
Although glaucomatous optic neuropathy is a disease in which
RGCs and their axons are lost progressively, other non-RGC retinal
neurons have been shown to be affected in a number of human
(Vaegan et al., 1995) and animal research (Bayer et al., 2001a,b;
Grozdanic et al., 2003a, 2004; Mittag et al., 2000; Nork et al.,
2000; Panda and Jonas, 1992a,b) studies. At present we have no
definite explanation for the pathogenesis leading to the degenerative functional and structural changes observed in the present
studies, and the mechanism by which outer, inner and innermost
retinal layers become compromised in the present model is not
clear, but may result from either or a combination of different types
of insults. In addition to the axotomy-like compressive damage to
RGC axons somewhere near the optic nerve head (Quigley and
Anderson, 1976, 1977; Salinas-Navarro et al., 2009a, 2010),
damage to non-RGC retinal neurons may also imply additional
damage to the retina, perhaps of ischemic nature. It has been largely
known that increased IOP may result in compromise to the inner
retinal blood supply, thus a vascular compromise or mechanical
compression of retinal vessels cannot be disregarded completely
(Costa et al., 2003; Flammer et al., 2002; Mozaffarieh et al., 2008).
Indeed, in addition to RGC loss (Lafuente López-Herrera et al., 2002),
permanent alterations of the a- and b-waves were observed in
studies in which transient ischemia of the retina for 90 min was
induced by a selective ligature of the ophthalmic vessels (AvilésTrigueros et al., 2003; Mayor-Torroglosa et al., 2005; Vidal-Sanz
et al., 2007). Moreover, there was a diminution in the overall
thickness of the retinal layers when examined in cross sections
(Avilés-trigueros et al., 2003; Mayor-Torroglosa et al., 2005) similar
to those described by others (Grozdanic et al., 2003a). Changes in
neurons different from RGCs were already evident 3 weeks after
lasering and the first remarkable signs were changes at the OPL
level with loss of bipolar cell terminal dendrites and synaptic
contacts between horizontal cells and photoreceptors. Loss of
photoreceptors was not evident at the first time points analyzed,
but by 9 weeks after lasering the number of photoreceptors per row
was almost halved to the normal value. Our present results show by
14 weeks changes in the overall retinal anatomy with some
remodeling signs, such as the mislocation of the horizontal cell
bodies. Indeed, secondary degeneration of the inner retina has been
described following degeneration of the outer retina (Villegas-Pérez
et al., 1996, 1998; Wang et al., 2000, 2003), and remodeling of retinal
layers is a common finding in retinal degeneration models (Cuenca
et al., 2004; Jones et al., 2003; Marc et al., 2003; Pignatelli et al.,
2004). Sprouting is frequent in retinal degeneration models, and
bipolar and horizontal cells try to find a presynaptic input at the
photoreceptor cell layer, and their dendrites grow looking for their
contacts. We have seen sprouting in other degeneration rodent
models such as the rd mouse (Rossi et al., 2003; Strettoi and
Pignatelli, 2000; Strettoi et al., 2003) or the RCS and P23H rat
(Cuenca et al., 2004, 2005a,b). This sprouting can be limited by some
therapeutic strategies, as cell-based therapy (Pinilla et al., 2007).
Finally, glial activation has also been an important contributor to the
283
OHT induced pathology that is also found in glaucomatous optic
neuropathy (Hernández et al., 2008; Woldemussie et al., 2004; Soto
et al., 2008; Marsh-Armstrong et al., 2010)
Acknowledgements
The authors would like to thank the technical contribution of
Isabel Cánovas, José M. Bernal and Leticia Nieto. This work was
supported by research grants from the Regional Government of
Murcia, Spanish Ministry of Science and Innovation and Fundaluce;
FIS PIO06/0780 (MPVP); 04446/GERM/07, SAF 2009-10385; RD07/
0062/0001 (MVS), FIS PI04/2399 (IP), BFU2009-07793/BFI, RD07/
0062/0012, Fundaluce, ONCE (NC), RD07/0062/0008 (PdlV).
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