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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 increaseof 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,*

aDepartamento de Fisiología, Genética y Microbiología, Universidad de Alicante, 03690 San Vicente del Raspeig, Alicante E-03080, SpainbHospital Miguel Servet, Zaragoza, Instituto Aragonés de Ciencias de la Salud, SpaincDepartamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, E-30100 Espinardo, Murcia, SpaindDepartamento de Fisiología, Facultad de Medicina, Universidad de Alcalá, E-28871 Alcalá de Henares, Spain

a r t i c l e i n f o

Article history:Received 11 February 2010Accepted in revised form 25 May 2010Available online 1 June 2010

Keywords:ocular hypertensionadult albino miceimmunohistochemistryERGphotoreceptorsbipolar cells

* Corresponding author. Tel.: þ34 868883961; fax:E-mail address: ofmmv01@um.es (M. Vidal-Sanz).

1 These authors contributed equally to this work.

0014-4835/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.exer.2010.05.020

a b s t r a c t

In adult albino mice the effects of increased intraocular pressure on the outer retina and its circuitry wasinvestigated at intervals ranging 3e14 weeks. Ocular hypertension (OHT) was induced by cauterizing thevessels 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 eachother prior to and at different survival intervals of 2, 8 or 12 weeks after lasering. Animals were processedat 3, 9 or 14 weeks after lasering, and radial sections were obtained in the cryostat and further processedfor 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 proteinbassoon, which labels photoreceptor ribbons and nuclei were identified using TO-PRO. Laser photoco-agulation of the perilimbar and episcleral veins of the left eye resulted in an increase in mean intraocularpressure to approximately over twice its baseline by 24 h that was maintained for approximately five daysreaching basal levels by 1 week. ERG recordings from the different groups of mice showed their a-, b-waveand 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 notdocument any changes in the outer nuclear layer (ONL) but both ON-rod bipolar and horizontal cells hadlost their dendritic processes in the outer plexiform layer (OPL). Sprouting of horizontal and bipolar cellprocesses were observed into the ONL. Fourteen weeks after lasering, protein kinase-C antibodiesshowed morphologic changes of ON-rod bipolar cells and calbindin staining showed abnormal horizontalcells and a loss of their relationship with their presynaptic input. Moreover, at this time, quantitativestudies indicate significant diminutions in the number of photoreceptor synaptic ribbons/100 mm, and inthe thickness of the outer nuclear and plexiform layer, when compared to their fellow eyes. Increasedintraocular pressure in Swiss mice results in permanent alterations of their full field ERG responses andin changes of the inner and outer retinal circuitries.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Previous studies on ocular hypertensive mice models havefocused predominantly on degenerative changes of the retinalganglion cell (RGC) population and their axons in the retinal fibre

þ34 868883962.

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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 thechanges at the RGC population, other retinal neuronal populationsmay also be affected (Bayer et al., 2001a,b; Mittag et al., 2000)including the inner (Panda and Jonas,1992a) and outer nuclear layersof the retina (Nork et al., 2000; Panda and Jonas, 1992b). Affectationof other non-RGC neuronal populations in the retina has been shownin a number of morphological and functional studies that have usedelectroretinogram (ERG) recordings and the measurement of the

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thickness of the inner andouter retinal layers (Fazio et al.,1986;Korthet al., 1994; Salinas-Navarro et al., 2009a). Experimental models ofelevated 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 causesretinal damage in adult albino mice we have used an ocularhypertension (OHT) induced retinal damage model, based on laserphotocoagulation of the perilimbal and episcleral veins (Salinas-Navarro et al., 2009a, 2010). A recent study has shown in adultalbino Swiss mice that lasering of the limbal tissues results in rapidelevation of the IOP which in turn results shortly after in diffuse aswell as focal loss of RGCs, that adopted the form of pie-shapedwedges with their vertex located towards the optic nerve, andexhibited variable sizes ranging from a small sector to severalquadrants of the retina (Salinas-Navarro et al., 2009a). Moreover,following elevated IOP there were permanent diminutions of thescotopic threshold response (STR) associated with the loss of thegreat majority of RGCs, indicating the value of noninvasive func-tional ERG losses associatedwith RGC loss in adult rodents (Alarcón-Martínez et al., 2009; Salinas-Navarro et al., 2009a). In addition therewere also permanent diminutions of the major components of theERG, the a- and b- waves, as well as lengthening of their implicittimes, all of which imply severe alterations of the inner and outernuclear layers of the retina (Salinas-Navarro et al., 2009a). In thepresent studies we have extended previous observations and havefurther studied short and long-term, qualitatively andquantitatively,functionally and morphologically, the retinal changes secondary toOHT in adult albino Swissmice, not only at the outer retinal neuronsbut also at the neurotransmitter level. We present evidence indi-cating that following laser injury to the aqueous outflowpathways inthe Swiss mice there is a functional impairment as well as retinaldegeneration that affects the inner and outer synapse and nuclearlayers, resulting in alterations of the inner and outer retinal circuit-ries. Short accounts of this work have been published in abstractform (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 malemice (40e45 g), obtained from the breeding colony of the Univer-sity of Murcia (Murcia, Spain). Mice were housed in temperatureand light controlled roomswith a 12 h light/dark cycle and had foodandwater ad libitum. Light intensity within the cages ranged from 9to 24 luxes. Animal manipulations followed institutional guide-lines, European Union regulations for the use of animals in researchand the ARVO statement for the use of animals in ophthalmicand vision research. All surgical manipulations were carried out, aspreviously described (Salinas-Navarro et al., 2009b,c), undergeneral anesthesia induced with an intraperitoneal (i.p.) injectionof a mixture of ketamine (75 mg/kg, Ketolar�, Parke-Davies, S.L.,Barcelona, Spain) and xylazine (10 mg/kg, Rompún�, Bayer, S.A.,Barcelona, Spain). At the end of the lasering treatment, retinalblood flow was assessed by examination of the eye fundus throughthe operating microscope (Vidal-Sanz et al., 2001, 2007; Avilés-Trigueros et al., 2003). During recovery from anaesthesia, micewere placed in their cages, and an ointment containing tobramycin(Tobrex�, Alcon S.A., Barcelona, Spain) was applied on the corneato prevent corneal desiccation. Additional measures were takento minimize discomfort and pain after surgery. Animals were

sacrificed with an i.p. injection of an overdose of pentobarbital(Dolethal Vetoquinol�, Especialidades Veterinarias, S.A., Alco-bendas, Madrid, Spain).

2.2. Induction of ocular hypertension

Ocular hypertension (OHT) was induced by cauterizing thevessels draining the anterior part of the mice eye, as recentlyreported (Salinas-Navarro et al., 2009a). In brief, on anesthetizedmice the left eyes were treated on a single session with a combina-tion of diode laser (532 nm, Quantel Medical, Clermont-Ferrand,France) burns. Laser beamwas directly deliveredwithout any lenses,aimed to the limbal and epiescleral veins. Special care was taken toavoid 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 Wrespectively. Mice received each approximately 72 spots, in a singlesession. Animals were housed in temperature and light controlledroomswith a 12 h light/dark cycle andhad food andwaterad libitum.

2.3. IOP measurement and experimental groups

The IOP was measured under anesthesia in both eyes witha rebound tonometer (Tono-Lab�, 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 timepoint, 36 consecutive readings were carried out for each eye,averaged and shown as means� SD for each time examined. Toavoid fluctuations of the IOP due to the circadian rhythm in albinoSwiss 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, becausegeneral anesthesia lowers IOP in rodents we measured the IOPtreated eye as well as the contralateral intact fellow eye in all theexperiments. Animals were divided into several groups sacrificed atincreasing survival intervals of 3 (group I, n¼ 5), 9 (group II, n¼ 4)or 14 (group III, n¼ 6) weeks after lasering.

2.4. ERG recordings

Prior to ERG recordings, animals were dark adapted overnightand their manipulation was done under dim red light (l> 600 nm),as recently described (Salinas-Navarro et al., 2009a). In brief, micewere anaesthetized, their IOP was measured as above, and bilateralpupil midriasis was induced by applying in both eyes a topical dropof 1% tropicamide (Tropicamida 1%�, Alcon-Cusí, S.A., El Masnou,Barcelona, Spain). The light stimulation device consisted in Ganzfelddome, which ensures a homogeneous illumination anywhere in theretina, with multiple reflections of the light generated by lightemitting diodes (LED), which provided a wide range of light inten-sities. For high intensity illuminations, a single LED placed close(1 mm) to the eyewas used. The recording systemwas composed byBurianeAllen bipolar electrodes (Hansen Labs, Coralville, IA, USA)with a corneal contact shape; a drop of methylcellulose 2%(Methocel 2%�; Novartis Laboratories CIBAVision, Annonay, France)was placed between the eye and the electrode to maximizeconductivity of the generated response. The reference electrode wasplaced in the mouth and the ground electrode in the tail. Electricalsignals generated in the retina were amplified (�1000) and filtered(band pass from1 Hz to 1000 Hz) by the use of commercial amplifier(Digitimer Ltd, Letchworth Garden City, UK). The recorded signalswere digitized (Power Lab; AD Instruments Pty. Ltd., Chalgrove, UK)and displayed on a PC computer. Bilateral ERG recording wereperformed simultaneously from both eyes.

Light stimuli were calibrated as follows before each experimentand the calibrationprotocol assured the same recordings parameters

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for both eyes. The stimuli device was formed by 3 independentcircuits of 4, 8 or 16 white LEDs, placed over the Ganzfeld dome. Theillumination was controlled by a computer which sent an electricalcurrent to one of the LED circuits. Before the actual experiment,the stimuli device was calibrated by a photometer situated insidethe Ganzfeld dome, in approximately the same position were theanimals headwas placed.We presented a series of stimuli (currents)for each LED-circuit, along a wide intensity range, and we obtainedthe corresponding photometer readings (cd/m2) for each stimulus.According to the time exposed to the stimulus presented to theanimal, we multipled the value of the light (cd/m2) by the stimulus’exposure time (s) and we obtained the logarithm of the result (logunit cds/m2).

The scotopic ERG responses were recorded by stimulating theretina with light intensities ranged between 10�5 and 10�4 cd sm�2

for the scotopic threshold response (STR),10�4 and 10�2 cd sm�2 forthe rodmediated response andbetween 10�2 and10�0.5 cd sm�2 forthe mixed (response mediated for rods and cones) response. Foreach light intensity, a series of ERG responses were averaged and theinterval between lightflasheswas adjusted to appropriate times thatallowed response recovering. At the end of each session the animalswere treated with topical tobramicine (Tobrex�, Alcon-Cusí, S.A., ElMasnou, Barcelona, Spain) in both eyes. The analysis of the differentrecordings was performed with the normalization criterions estab-lished for the ISCEV for the measures of the amplitude and implicittime of the different waves which were studied (Salinas-Navarroet al., 2009a). ERG were recorded prior to lasering and at 2, 8 or 12weeks after lasering for groups I (n¼ 5), II (n¼ 4) or III (n¼ 6),respectively.

The a-wavewasmeasured from the baseline to the first valley, ca.10 ms, from theflash onset and the b-wave amplitudewasmeasuredfrom the bottom of the a-wave trough to the top of the hill of thepositive deflection; the time point of the b-wave measurement,varied depending of the intensity used. The implicit time wasmeasured from the presentation of the stimulus to the peak of the b-wave. Data from operated and non-operated eyes were compared;ERG wave amplitudes and implicit times were calculated for eachanimal group and thepercentage of difference between the operatedand the non-operated eyes were obtained for each stimulus andfurther averaged (mean� SEM). The results were analyzed withSigmaStat� 3.1 for Windows� (Systat Software, Inc., Richmond, CA,USA). Descriptive statistics were calculated and t-test was used in allstudied groups for the comparison between prior and post lasering.We performed the t-test prior to surgery to compare the response ofboth eyes to demonstrate similar functionality in both eyes prior tothe lesion. The statistic significance was placed in a p< 0.05 for alltests 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 eyecupswere fixed in 4% paraformaldehyde in 0.1 M PBS at pH 7.4 for 1 hand then washed in 0.1 M PBS before being cryoprotected in 15%sucrose for 1 h, 20% sucrose for 90 min and 30% sucrose overnightat 4 �C. Next day they were embedded in OCT and cross sections ofthe retina were cut at 16 mm thickness on a cryostat in a horizontalplane, and mounted on glass slides. Sections were treated as inprevious studies (Cuenca et al., 2002, 2004, 2005a,b) for immu-nostaining, using as primary antibodies mouse anti-recoverin1:1000 (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 ribbonswere identified using an antibody against the protein bassoon,which labels photoreceptor ribbons (mouse anti-bassoon 1:5000;

Stressgen). Nuclei were identified using TO-PRO 1:1000 (MolecularProbes). Slides were mounted in watermount (Vector Labs) andcoverslipped for viewing by confocal microscopy (Leyca TCS SP2).To control for non-specific staining, some sections were stainedomitting the primary antibody.

Qualitative examination of the sections revealed differencesbetween injured and control retinas that were more marked in thegroup of animals examined 14 weeks after laser, thus for this groupwe have quantified in a masked fashion: i) the thickness of the outernuclear layer (ONL) and outer plexiforme layer (OPL) using a TO-PROstain which labels nucleus of retinal cells, and ii) the numberof synaptic contacts between photoreceptors and bipolar cells, bycounting the number of spots bassoon immunopositive. Bassoonlabels the synaptic ribbon at the axon terminal of photoreceptors,and each bassoon spot in the OPL is associated with its corre-spondingbipolar dendrite tip pairing. Cross sections from the centralretina, close to the optic nerve head, in 5 animals, were photo-graphed. At least seven pictures from different alternate sections at63� ofmagnification for TO-PRO images, and 100� of magnificationfor bassoon immunostained retinas, were obtained from eachexperimental and its corresponding right (non-lasered) fellow eye,which was used as a control. The number of spots bassoon positivewas expressed as a mean of labeled cells per 100 mm. The thicknessof theONL andOPLweremeasured using ImageJ NIHprogramand atleast thirty linearmeasureswere done for each picture. For statisticalanalysis, 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 valuesregistered from the lasered-eyes, within each of the animals andthe subgroups processed at different survival intervals, but ingeneral the results were rather consistent (Fig. 1AeC).

Overall our data show a similar time course elevation of the IOPfor all groups of mice analyzed (Fig. 1AeC). There was an importantincrease of the IOP by 24 h after lasering, to twice its baseline valuethat remained elevated for the following four days and returned tobaseline values by one week after lasering. Indeed, the statisticalanalysis showed that the IOP values were comparable among thedifferent groups and eyes (KurskaleWallis test, p¼ 0.3547). Twentyfour hours after lasering, the IOP values were comparable withinthe different animal groups in the lasered (KruskaleWallis test,p¼ 0.2546) as well as in the contralateral uninjured right eyes(KruskaleWallis test, p¼ 0.5827). Therewas a substantial increase ofthe IOP thatwas already evident 24 h after lasering (ManneWhitneytest, p¼ 0,0011) and was maintained up to the fifth day (Man-neWhitney test, p¼ 0,0011), returning to their basal values by oneweek after lasering when the IOP values in animals of the differentgroups were comparable for both eyes (KruskaleWallis test,p¼ 0.1859) (Fig. 1C).

3.2. ERG responses

3.2.1. ERGs in control albino miceTo study the effect of elevation of the IOP on the ERG waves in

albino mice, simultaneous ERG recordings were performed fromright and left eyes of each animal prior to surgery. No significantdifferences were observed in the a- or b-wave ERG amplitudes,between the left and right eyes in any of the animals of this studyprior to surgery (Fig. 2A). Representative examples of the ERGtraces recorded in both eyes, prior to surgery, in response to flashstimuli of increasing intensity are shown in Fig. 3. No ERG a-wave

Fig. 1. Intraocular pressure measurements in adult albino mice. AeC Histogram showsmean (�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 14weeks, respectively, after laser photocoagulation of the limbal and episcleral veins ofthe left eye. Each received an average of 72 laser-burning spots in a single session. IOPvalues in group I (A) rose by day 1 post-treatment to twice their normal values andreturned to basal preoperative levels by 1 week. IOP values in group II (B) followeda similar pattern of pressure variations. IOP values in group III (C) also showed by 1da raise to twice their normal values, that were maintained for the following four daysand decreased gradually to basal levels by one week, remaining at basal levels for therest of the study. Thus, IOP elevation in all these three groups was not maintainedbeyond 1 week.

Fig. 2. Electroretinographic amplitude measurements in albino mice. Bar histogramshowing averaged data (mean� SEM) for the right and left eye a-wave and b-waveERG amplitudes versus stimulus intensities from the groups of mice studied. A showsa- 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 andD show a- and b-wave ERG amplitudes at 2, 8 or 12 weeks after lasering respectively.

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was observed for light intensities below�2 log cds/m2. The b-waveelicited by light intensities from�3.66 to�0.5 log cds/m2 increasedexponentially (Fig. 3).

3.2.2. ERGs in experimental albino mice after elevated IOPERG recordings were performed simultaneously from right non-

treated and left lasered eyes in albino mice at specific survivalintervals after lasering. A representative example from group I miceregistered 2 weeks after lasering shows the STR and scotopic a- and

Fig. 3. Scotopic electroretinographic recordings in albino mice. Examples of the ERGtraces 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 lightintensities of �4.26 log cds/m2 and rod and mixed responses were elicited by lightintensities from �3.66 to �0.59 log cds/m2. Vertical arrows denote presentation oflight stimulus. No significant difference in the ERG amplitudes between left and righteyes were observed prior to laser (T-test, p> 0.05). However, at 2 week after laseringthe limbal tissues, there is a clear reduction in all studied waves from the left eye(T-test, p< 0.001).

Fig. 4. Changes in ERG measurements at different survival intervals. Histogram plotsillustrating changes in the amplitudes of the ERG components studied (a-, b-waves andpSTR), recorded from the experimental groups analyzed 2, 8 or 12 weeks after lasering.Measurements for all showed light intensity stimulus were averaged (mean� SEM) forthe a-, b-waves and pSTR, and presented as % of their fellow eyes. For the groupsstudied at 2, 8 or 12 weeks after laser treatment, the a-, b-wave and pSTR amplitudewere 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.

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b-waves siginificantly reduced when compared to the pre-laseringrecordings (Fig. 3). In addition, data are shown as averages ofabsolute wave amplitudes (mean� SEM) in lasered and contralat-eral fellow eyes (Fig. 2BeD). ERG recordings from the differentgroups of mice showed average reductions in the scotopic andmixed ERG recorded at all survival intervals examined. Their a-,b-wave and pSTR amplitudes, when compared to their contralateralfellow 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 12weeks after lasering, respectively (Fig. 4). These values weresignificantly 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 weeksafter lasering, and persisted for up to 12 weeks, the longest timeinterval recorded in the present studies, indicating that thesefunctional alterations were permanent.

3.3. Immunofluorescence findings

3.3.1. Three weeks after acute IOP increasePhotoreceptors were identified using recoverin (McGinnis et al.,

1999) and their nuclei were stained using TO-PRO. Three weeksafter lasering no remarkable changes were found at the photore-ceptor level. Photoreceptor nuclei labeled with TO-PRO maintaineda similar number of rows, although some of the cell bodies located at

the inner part of the outer nuclear layer (ONL) had lost their align-ment andwere lightlymisplaced 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 coneswe observed that transducin immunoreactivity in normal micewas particularly prominent in the cone outer segments, cone innersegments and cell bodies, while the pedicles were more lightlystained. There were some subtle differences in cone morphologybetween the lasered andcontrol eyes (Fig. 5CandD). In theuntreatedmouse eyes, cone cell bodies stained with transducinwere confinedto the outer third of the ONL,whereas in the lasered eyes some of thecone cell bodies were displaced towards the middle portion of theONL (Fig. 5D, arrows). In most studied retinas no differences werefound in the cone outer segment length and morphology. However,the shape of cone pedicles in the lasered eyes looks somewhat flat-tened when compared to control retinas (Fig. 5C, D).

To study if the IOP increase induces retinal changes at the OPLlevel, we stainedON rod bipolar cells using antibodies against PKC-a.At this time point, bipolar cell dendrites showed the first signs ofOHT-induced retinal damage; therewas a loss of the apical dendritesand their dendritic terminals appeared to be reduced in length andin 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 photore-ceptors at the OPL level are the horizontal cells. Horizontal cells canbe identified using antibodies against calbindin. In normal mouseretinae, the dendrites of the single horizontal cell type, the B-typecell, contact cone terminals, and the axon terminal contacts rodterminals. In the lasered eyes, changes were also remarkable at thehorizontal cells. The regular and dense plexus of horizontal cellsprocesses in the OPL was lost and there was a clear reduction ofdendrites and axon terminal tips (Fig. 5G and H). In the retinas ofthe treated eyes, horizontal cell processes showed clear sproutingand some of their terminal ends were found inside the ONL (Fig. 5Harrows).

3.3.2. Nine weeks after laseringNine weeks after lasering the number of photoreceptor rows, as

seen with TO-PRO and recoverin, were diminished. The decreasewas obvious comparing the ONL thickness of the normal mice andthe lasered mice at this time point (control versus the lasered eye)

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 inretinal 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 conemorphological 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) PKC-a 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).

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(Fig. 6A and E versus 6B and F). In the untreated eyes of the normalmice the staining with recoverin and TO-PRO showed 12- to 13-photoreceptor rows forming the ONL while in the laser-treated eyethere was an average of 8e9 photoreceptor rows. Rod bipolar celldendrites immunostained with antibodies against PKC-a werediminished and showed less complex branching patterns in treatedeyes (Fig. 6D) compared with control ones (Fig. 6C). In the retinas of

the lasered eyes, some sprouting of rod bipolar cells dendrites couldbe observed at the ONL level (Fig. 6D, arrows). In addition tophotoreceptor staining, recoverin antibodies immunostained twotypes of cone bipolar cells the ON-cone bipolar Type 8 and OFF-cone bipolar Type 2; both of them showed a decrease of immu-noreactivity in treated eyes (Fig. 6F) compared to untreated eyes(Fig. 6E).

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 theONL. 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 PKC-a. 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 somesprouting 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) andphotoreceptor and cone bipolar cells labelled with antibody against recoverin (red). There was a loss of horizontal cell processes and their contacts with photoreceptors terminals intreated 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 bipolarcells at the INL was observed compared with control retina (E). Scale bar represents A, B, E, F¼ 20 mm, C, D¼ 10 mm.

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Nine weeks after lasering, horizontal cell dendrites showeda decrease of processes in the OPL and some sprouting into the ONL(Fig. 6F, arrow in inset). At this time no major changes were foundat the inner retina, the inner plexiform layer (IPL) thicknessremained normal and the three plexus stained with antibodiesagainst calbindin could be identified.

3.3.3. Fourteen weeks after laseringA clear reduction of neurons in the ganglion cell layerwere found

in treated eyes stained with TO-PRO (Fig. 7C and D) compared withnon treated eyes (Fig. 7A and B). The same reduction of neurons inthe ganglion cell layer was observed when the retinas were stainedwith antibodies against calbindin (Fig. 7A and C, arrows).

Fourteen weeks after lasering TO-PRO staining showed a cleardiminution in the number of photoreceptors (Fig. 7A and C).The thickness of photoreceptor layer was markedly reduced in sizewhen compared to the untreated eye (compare Fig. 7D versus 7Band see below).

In laser-treated mouse retinas, ON rod bipolar cells dendriticbranches (Fig. 8B) were less profuse and some cells were devoid ofdendrites compared with control retinas (Fig. 8A). An obviousdecrease of cell number, immunoreactivity and dendritic terminalswere found in treated eyes (Fig. 8B). A double immunolabelling withthe antibody against the presynaptic protein bassoon which labelsphotoreceptor synaptic ribbon terminals in rod spherules and cone

Fig. 7. Low magnification cross section retinas 14 weeks after lasering, double labeled withwere found comparing control (A,B) and treated eyes (C,D). Significant ganglion cells losImmunopositive calbindin ganglion cells were decreased in treated eyes (C arrows) versus cocell layer. Scale bar¼ A and C 200 mm, B and D¼ 20 mm.

pedicles in the OPL allowed examining their synaptic contact distri-bution with ON rod bipolar cells immunostained with antibodiesagainst PKC-a (Fig. 8A andB). In the retina of the control eyes, ON rodbipolar cell dendrite tips (green) were all associated with horseshoes-like bassoon immunoreactivity (red) (Fig. 8A). In the laseredeyes 14 weeks after the treatment, the more remarkable changewasthe loss of bipolar cell dendrites and a diminution in the number ofbassoon immunoreactive spots; both findings suggest a loss ofcontact betweenONrodbipolar cell dendrites and the photoreceptorsynaptic axon terminals. Some bassoon immunoreactive spots werefound at the ONL layer with no bipolar cell contacts (Fig. 8B).

This reduction was associated with a decrease of the OPLthickness (Figs. 8A, B and 9A). In order to assess if this change wasdue to the loss of the photoreceptor axon terminals we carried outa double staining for calbindin and synaptophysin (SYP), a synaptic-vesicle protein present in the photoreceptor axon terminal that wasused to identify the presynaptic elements of pedicles and spherulesof their respective photoreceptor types. A close relationshipwas found at the OPL between SYP photoreceptor axon endings andhorizontal cells terminals (Fig. 8C). In normal retinae, the dendritictips of the horizontal cells (labeled with calbindin) were associatedwith rod spherules (labeled with SYP). SYP labeling was continu-ously distributed across the OPL (Fig. 8C). In the retina of the treatedeyes, there was a dramatic reduction of SYN immunoreactivity(Fig. 8D) showing a great loss of photoreceptor axon terminals that

TO-PRO and antibodies against calbindin (green). A reduction of photoreceptor rowss labeled with TO-PRO were also observed comparing control B and treated eyes D.ntrol retina (A arrows). ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion

Fig. 8. Confocalfluorescencemicrographs of retinal cross sections showing synaptic contacts between bipolar andhorizontal cellswithphotoreceptors in theOPL 14weeks after lasering(B,D,F) comparedwith control retinas (A,C,E). (A and B) Confocal fluorescencemicrographs of a retinal cross section showing a loss of photoreceptor ribbon synapseswith ON rod bipolarcell at the OPL level in the retina of treated eyes (B). The synaptic ribbonswere visualized with an antibody against the protein bassoon (red) which labels photoreceptor ribbons in bothcone pedicles and rod spherules. Bipolar dendrites were labeledwith an antibody against PKC-a. In treated eyes bipolar cell (B) dendritic branches were less profuse and some cells weredevoid of their dendrites. A few contacts between bipolar terminals and photoreceptor axon terminals immunostained with bassoon (red) were observed. (C and D) Horizontal celldendrites stainedwith antibodies against calbindin andphotoreceptor axon terminal immunostainingwith antibodies against synaptophysin. In control animal, using SYN staining, 3 to 4axon 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 andusing 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-F10 mm.

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were missing their contacts with the few remained horizontal cellsterminal tips (Fig. 8D, F).

Using a TO-PRO stain the mean thickness of the outer nuclearlayer (ONL) was 37.24�1.74 mm (mean� SEM; n¼ 5) for the OHTeyes and 45.88� 6.10 mm (n¼ 5) for the fellow eyes (Fig. 9A).Similarly the mean thickness of the outer plexiform layer (OPL) was7.52�1.48 mm (n¼ 5) for the OHT eyes and 10.58� 0.81 mm (n¼ 5)for the fellow eyes (Fig. 9B). There were significant differences forboth measurements (Unpaired t test, p¼ 0.0159 and p¼ 0.0038 forONL and OPL, respectively).

The number of synaptic contacts between photoreceptors andbipolar cells were compared in treated and fellow eyes by countingthe number of bassoon immunoreactive spots, expressed as a meanof labeled cells per 100 mm. This measurement was significantlygreater (Unpaired t test, p¼ 0.0060) in the control 82.65�10.61 mm(mean� SEM; n¼ 5) than in the OHT eyes 62.76� 5.59 mm (n¼ 5)(Fig. 9C).

4. Discussion

Various types of retinal injury have been proposed to study adultrodent RGCs in an effort to correlate their findings with the humanglaucomatous 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), transientischemia 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-Navarroet al., 2009a, 2010). Because, one of the most important riskfactors 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 muchattention from investigators in an effort to correlate their findingswith human glaucoma, and these models have proven useful toprogress our understanding of OHT-induced RGC and optic nerve

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 felloweyes, 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 laser-treated 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.

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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 andSretavan, 2010; Grozdanic et al., 2003b; Holcombe et al., 2008; Jiet al., 2005; Mabuchi et al., 2004; Salinas-Navarro et al., 2009a).However, much less information is available regarding the func-tional, structural and molecular changes secondary to OHT, and inparticular this information is lacking in the adult albino Swiss mice.In the present studies, using electrophysiological and immunoflu-orescence techniques we have further examined from 3 to 14weeks, the effects of ocular hypertension (OHT) on the function andstructure of the adult albino Swiss mice retina. Our functionalresults show that there aremajor reductions in the amplitudes of a-,b-wave and pSTR, which persist for as long as 12 weeks, indicatinga permanent damage to the outer and inner nuclear layers.Morphologically, we observed changes at the level of the OPL thatappear as early as 3 weeks, progressed with time and by 14 weekstherewas loss of photoreceptors, displacement of other cell types ashorizontal cells, and a diminished relationship between photore-ceptors and horizontal cells. Moreover, at this time quantitativeanalysis of our morphological data (Fig. 9) indicate significantdiminutions in the number of photoreceptor synaptic ribbons andin the thickness of the ONL and OPL. Altogether the morphologicaldata could justify the permanent functional impairment observedin the ERG recordings.

In agreement with our recent report (Salinas-Navarro et al.,2009a) the IOP raised up to approximately over twice their basalvalueswithin 24 h, remained elevated for the following 4 days and atone week the IOP values returned to basal levels. Variability of theIOP is a common finding in rodent models of glaucoma (Morrisonet al., 1997, 2005, 2008) because different lasering methods resultin different IOP elevation profiles with variations in the peak IOPvalue, its latency and total duration of theOHT (Chauhan et al., 2002;Danias et al., 2006; Levkovitch-Verbin et al., 2002). Similar abruptelevations of the IOPwere observed for pigmentedmice inwhich thetrabecularmeshwork and the episcleral veins were photocoagulatedwith laser (Grozdanic et al., 2003a, 2004). However the pigmentedmice of Grozdanic and colleages (2004) showed persistent IOPincreases for over 30 days, while in our study the IOP elevation was

restricted to the first week. Thus, our present studymay be regardedas an acute or subacute OHT model, similar to a recent report inalbino mice (Fu and Sretavan, 2010), and this may be seen asa disadvantage when compared to a more chronic model of IOPelevation. Nevertheless our IOP profile produced a severe injury tothe retina that resulted in a number of features, such as sectorial lossof RGCs and degeneration of the nerve fiber layer (Salinas-Navarroet al., 2009a) that are typically found in an inherited mice modelof ocular hypertension (Buckingham et al., 2008; Jakobs et al., 2005;Schlamp et al., 2006; Soto et al., 2008) thus arguing in favour ofa similar damaging mechanism. Moreover, our present studieshighlight the fact that only long-term studies may show the fullconsequences of ocular hypertension, even when this was inducedfor a short period of time, as in the present studies. Indeed, many ofthe previous reports are short-term OHT studies, and this mightexplainwhy they did notfind evidence of photoreceptor or any othernon-RGC retinal damage.

In this studywehave focused on the OHT induced changes in nonretinal ganglion cell neurons, and thus we have studied functionallyandmorphologically the synaptic and nuclear inner and outer layersof the retina. In our present experiments analyzed at 2, 8 or 12weeks, we found that shortly after elevation of the IOP, by 2 weeks,therewas an alteration in the amplitudes of the a-, b-waves andpSTRof the ERG, and these results are analogous to those recentlyreported in a parallel study (Salinas-Navarro et al., 2009a) in whichERG were recorded up to 8 weeks. Our present results are also inagreement with those registered up to 30e40 days in OHT studies inpigmented mice (Grozdanic et al., 2003a,b, 2004; Holcombe et al.,2008) and with those registered 5 weeks after acute elevation ofthe IOP (Fortune et al., 2004). Moreover, our findings correlate wellwith the overall anatomical and structural changes found in a DBA/2NNiamice study showing a progressive thinningof theouter retinallayers and corresponding decreases of the a- and b-wave amplitudesin the ERG (Bayer et al., 2001a,b; Mittag et al., 2000). Other studieshowever, have reported reversible changes in a- and b- waveamplitudeswhen IOPvalues returned to basal levels (He et al., 2006).More recent studies, using acute IOP increases indicate a thresholdfor retinal damage resulting inpermanent alterations of the a- andb-waves after a substantial increase of the IOP (above 30e70 mm Hg)or when the cumulative time-IOP elevation integral is severe (Buiet al., 2005; Fortune et al., 2004; He et al., 2006; Kong et al., 2009).In our study the IOP normalizedwithin aweek after lasering, but the

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ERGwaves persistedwith diminished values for as long as 12weeks,thus it is possible that in our experiments, the IOP profile resulted insevere retinal damage that affected not only the RGC population andtheir axons as recently reported (Salinas-Navarro et al., 2009a, 2010),but also the inner and outer retinal layers, which are responsible forthe genesis of the a- and b-waves. Indeed, the ERG b-wave has beenwidely used to diagnose various types of retinal degenerationrelated to photoreceptor and/or inner retinal cells in animal studies(Peachey and Ball, 2003) and its amplitude correlates directlywith ON-bipolar activity (Stockton and Slaughter, 1989; Tian andSlaughter, 1995) and as such is most likely, in cases of photore-ceptor dysfunction, to be directly related to the level of transmissionfrom photoreceptors to ON bipolar cells via mGluR6 receptors.

Although glaucomatous optic neuropathy is a disease in whichRGCs and their axons are lost progressively, other non-RGC retinalneurons 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 nodefinite explanation for the pathogenesis leading to the degenera-tive functional and structural changes observed in the presentstudies, and the mechanism by which outer, inner and innermostretinal layers become compromised in the present model is notclear, but may result from either or a combination of different typesof insults. In addition to the axotomy-like compressive damage toRGC axons somewhere near the optic nerve head (Quigley andAnderson, 1976, 1977; Salinas-Navarro et al., 2009a, 2010),damage to non-RGC retinal neurons may also imply additionaldamage to the retina, perhaps of ischemic nature. It has been largelyknown that increased IOP may result in compromise to the innerretinal blood supply, thus a vascular compromise or mechanicalcompression 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 instudies in which transient ischemia of the retina for 90 min wasinduced by a selective ligature of the ophthalmic vessels (Avilés-Trigueros et al., 2003; Mayor-Torroglosa et al., 2005; Vidal-Sanzet al., 2007). Moreover, there was a diminution in the overallthickness of the retinal layers when examined in cross sections(Avilés-trigueros et al., 2003; Mayor-Torroglosa et al., 2005) similarto those described by others (Grozdanic et al., 2003a). Changes inneurons different from RGCs were already evident 3 weeks afterlasering and the first remarkable signs were changes at the OPLlevel with loss of bipolar cell terminal dendrites and synapticcontacts between horizontal cells and photoreceptors. Loss ofphotoreceptors was not evident at the first time points analyzed,but by 9weeks after lasering the number of photoreceptors per rowwas almost halved to the normal value. Our present results show by14 weeks changes in the overall retinal anatomy with someremodeling signs, such as the mislocation of the horizontal cellbodies. Indeed, secondary degeneration of the inner retina has beendescribed following degeneration of the outer retina (Villegas-Pérezet al.,1996,1998;Wang et al., 2000, 2003), and remodeling of retinallayers is a common finding in retinal degeneration models (Cuencaet al., 2004; Jones et al., 2003; Marc et al., 2003; Pignatelli et al.,2004). Sprouting is frequent in retinal degeneration models, andbipolar and horizontal cells try to find a presynaptic input at thephotoreceptor cell layer, and their dendrites grow looking for theircontacts. We have seen sprouting in other degeneration rodentmodels such as the rd mouse (Rossi et al., 2003; Strettoi andPignatelli, 2000; Strettoi et al., 2003) or the RCS and P23H rat(Cuenca et al., 2004, 2005a,b). This sprouting can be limited by sometherapeutic strategies, as cell-based therapy (Pinilla et al., 2007).Finally, glial activation has also been an important contributor to the

OHT induced pathology that is also found in glaucomatous opticneuropathy (Hernández et al., 2008;Woldemussie et al., 2004; Sotoet al., 2008; Marsh-Armstrong et al., 2010)

Acknowledgements

The authors would like to thank the technical contribution ofIsabel Cánovas, José M. Bernal and Leticia Nieto. This work wassupported by research grants from the Regional Government ofMurcia, 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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