Changes in visual fields and lateral geniculate nucleus in monkey laser-induced high intraocular pressure model

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Experimental Eye Research 86 (2008) 770e782 www.elsevier.com/locate/yexer

Changes in visual fields and lateral geniculate nucleus in monkey laser-induced high intraocular pressure model Masaaki Sasaoka a,b, Katsuki Nakamura a,c, Masamitsu Shimazawa d, Yasushi Ito d, Makoto Araie e, Hideaki Hara d,* a

b

Department of Behavioral and Brain Sciences, Primate Research Institute, Kyoto University, 41-2 Kanrin, Inuyama, Aichi 484-8506, Japan Advanced Medical Technology Unit, Research and Development Center, Santen Pharmaceutical Co. Ltd., 8916-16 Takayama-cho, Ikoma, Nara 630-0101, Japan c Department of Animal Models for Human Disease, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan d Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan e Department of Ophthalmology, University of Tokyo School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Received 26 November 2007; accepted in revised form 15 February 2008 Available online 26 February 2008

Abstract Monkey eyes are useful for ophthalmologic research into eye diseases because their histological and functional properties are very similar to those of humans. The monkey laser-induced high intraocular pressure (IOP) model is a common model for ophthalmologic research, especially into glaucoma. Although several studies using this model have focused on changes in visual field, retinal ganglion cells (RGC), and lateral geniculate nucleus (LGN), clear relationships among these changes in one and the same monkey have not been established. We therefore examined visual field changes, RGC and LGN numbers, and glial fibrous acidic protein (GFAP) immunohistochemistry in the LGN in each of two monkeys. Visual field sensitivity, RGC number, and neuronal density of LGN were all decreased by high IOP. The relationship between loss of RGC and decrease in visual field sensitivity depended on the eccentricity from the fovea. Moreover, LGN immunohistochemistry revealed greater increases in GFAP expression in the layers receiving a neuronal input from the high IOP eye than in those receiving a neuronal input from the contralateral untreated eye. From these results, we suggest that glaucoma may lead to changes in glial function not only in the retina, but also in the visual pathway, and that such central nervous system changes may be a hallmark of neuropathy in glaucoma, as in other neurodegenerative diseases. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: perimetry; glaucoma model; retina; lateral geniculate nucleus; glial fibrous acidic protein; monkey

1. Introduction Glaucoma is a progressive disease involving typical visual field defects associated with the death of retinal ganglion cells (RGC). These changes are thought to have several causes, including high intraocular pressure (IOP) (Gordon et al., 2002; Kass et al., 2002; Sommer et al., 1991), circulatory disturbances (Flammer et al., 2002; Osborne et al., 1999; Yamazaki and Drance, 1997), and an excess of excitatory

* Corresponding author. Tel./fax: þ81 58 237 8596. E-mail address: [email protected] (H. Hara). 0014-4835/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2008.02.004

amino acids (Dreyer et al., 1996; Osborne et al., 1999). In the clinical situation, procedures exist for the diagnosis of glaucoma and for the assessment of its progression, and many studies have reported relationships between changes in the visual field and other surrogate parameters, such as nerve fiber-thickness measurements and optic nerve head tomography (Bowd et al., 2001; Medeiros et al., 2004; Schuman et al., 1995; Zangwill et al., 2001). Although knowledge of the changes in the visual pathway [including the retina, lateral geniculate nucleus (LGN), and visual cortex] are important for our understanding of the pathophysiology of glaucoma, it is difficult to research these changes and their relationships in humans. Consequently, there have been only a few reports

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on subjects from different backgrounds (Harwerth and Quigley, 2006; Kerrigan-Baumrind et al., 2000) and one case report (Gupta et al., 2006). Many studies have tried to clarify the changes in the visual pathway occurring in glaucoma using a high IOP model in monkeys. Using the perimetry system for monkeys, reports have been made of the relationship between visual field sensitivity and RGC number (Harwerth et al., 1999, 2002, 2004), electroretinogram (Frishman et al., 1996, 2000; Harwerth et al., 2002), and cytochrome oxidase reactivity in LGN or visual cortex (Crawford et al., 2000, 2001; Harwerth et al., 2002). These reports have been useful for our understanding of the changes in each section of the visual pathway: for example, RGC degeneration has been shown to occur prior to visual field sensitivity changes or to certain metabolic changes in LGN and visual cortex in high IOP monkeys. However, an important omission from these previous studies is that the relationships among changes in visual field, RGC, and LGN in one and the same monkey have not been established. Glial cells have been reported to be important for the maintenance of neuronal activity within the central nervous system (CNS) (Haydon, 2001). Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is expressed within astrocytes in the CNS (Eng et al., 2000; Pekny and Pekna, 2004), and an upregulation of GFAP is a hallmark of CNS injuries such as Alzheimer’s disease and CreutzfeldteJacob disease (Eng et al., 2000). However, while the expression pattern of GFAP has been well studied, its role has not been clearly defined. Although an analysis of GFAP-knockout mice has shown them to be normal with regard to life-span, reproduction, and gross morphology (Gomi et al., 1995; Pekny et al., 1995), another report suggested that their astrocytes have shorter processes than normal and that their blood-brain barrier is impaired (Liedtke et al., 1996). Furthermore, GFAP-knockout mice have been observed to be more sensitive to a number of neuronal injuries (Nawashiro et al., 1998, 2000; Otani et al., 2006) even though synaptic functions are impaired by increased GFAP expression (Finch, 2003; Menet et al., 2001). Thus, as indicated above, the functions of GFAP are still enigmatic. In the retina, GFAP is also expressed within astrocytes in normal subjects, and its retinal expression is increased in astrocytes and Mu¨ller cells in several pathological conditions [e.g., in glaucoma patients (Tezel et al., 2003; Wang et al., 2002), in high IOP models in monkeys (Tanihara et al., 1997), rats (Ju et al., 2006; Kanamori et al., 2005; Wang et al., 2000; Xue et al., 2006), and mice (Inman and Horner, 2007), and in a rat excitotoxic injury model (Honjo et al., 2000)]. However, although GFAP expression in the retina has been well studied, there have been no reports concerning its expression in LGN after high IOP in monkeys. Thus, in the present study, we investigated the relationship between visual sensitivity and RGC number in two individual high IOP monkeys, and we also examined histological and GFAP immunoreactivity changes in LGN in the same monkeys to examine the role of GFAP in glaucomatous visual field changes.

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2. Materials and methods 2.1. Animals Two experimentally naive male cynomolgus monkeys [Monkeys A and B; Macaca fascicularis, 5 years old, weighing 4.0 kg (A) and 4.5 kg (B)] were used as subjects. Each monkey was housed in an individual cage in a monkey colony. Ophthalmoscopy and refractometry conducted before the experiment revealed no ocular abnormalities or high ametropia in either of the monkeys. Each monkey was deprived of water before each daily session, and fruit juice was given as reward during the experiment. Supplemental water and vegetables were given after the session when needed. Food (monkey chow) was continuously available in the cage. All animal care and experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Research Council (1996), and with the Guide for the Care and Use of Laboratory Primates published by Kyoto University Primate Research Institute (1986 and 2002). 2.2. Induction of experimental glaucoma An elevation of intraocular pressure (IOP) was induced by applying argon blue/green laser photocoagulation burns to the trabecular meshwork of the right eye of each monkey, with the left eye being used as an untreated control, as previously described (Quigley and Hohman, 1983). For the laser treatment, the animals were anesthetized with an intramuscular injection of ketamine (8.75 mg/kg; Ketalar 50Ò, Sankyo, Tokyo, Japan) plus xylazine (0.5 mg/kg; Celactal, Bayer Yakuhin Ltd., Tokyo, Japan). A single-mirror Goldmann lens filled with a physiological solution (ScopisolÒ15; Senjyu Pharmaceutical, Osaka, Japan) was placed on the eye to be treated. The argon laser was focused on the mid-portion of the trabecular meshwork, and a total of 150 laser-beam spots were applied around 360 (spot size 100 mm; power 1000 mW; exposure time 0.2 s) using an argon laser photo-coagulator (Ultima 2000 SEÒ; Coherent Inc., CA, USA) attached to a standard slit-lamp microscope (BQ 900; Haag-Streit, Ko¨niz, Switzerland). Two weeks after the first treatment, the laser treatment was repeated to produce a maintained elevation of IOP. 2.3. Visual field testing We measured each monkey’s visual field as follows, using a procedure described previously in detail (Sasaoka et al., 2005). During the experimental session, the monkey was seated in a primate chair (with its head fixed by a headrestraining device) in a dimly lit, soundproofed, experimental room. A 22-in monochrome CRT monitor (ME224f; maximum resolution 1600  1200; Totoku Electric, Tokyo, Japan) was placed 30 cm from the monkey’s eyes. The brightness of the CRT monitor was calibrated regularly using a luminance colorimeter (BM-7; Topcon, Tokyo, Japan). Visual stimuli were presented on the CRT monitor, which subtended 67  52 (width  height) at the 30-cm viewing distance.

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While the visual field of one eye was being measured, the fellow eye was covered with a soft eye-patch. The monkey was trained to perform a visual reaction-time task by pressing and releasing a lever to get a juice reward. We measured each monkey’s visual field twice a week from before the induction of high IOP to 15 weeks after the first laser application. Before the visual field testing, each monkey’s gaze was calibrated. To measure the monkey’s eye position, two camera sets [each consisting of a CCD (charge-coupled device) camera (XC-75; Sony, Tokyo, Japan) and an infrared irradiation device] were placed inside the experimental room. An image of the monkey’s pupil was taken by the CCD camera, then digitized and transferred to a personal computer. From the eye image, the pupil was delineated on the basis of the luminance difference, and the center of an oval approximately fitted to the pupil was calculated. At the beginning of each daily session, the eye-measuring system was calibrated using 9 fixation point locations in the visual reaction-time task. Using this 9-position data, we computed the center of the eyeball and the vector from the eyeball’s center to each of the 9 positions. Thus, the monkey’s eye position was computed by transforming the coordinates of the center of the eye image into a vector of the eye direction (Matsuda, 1996; Sato and Nakamura, 2001, 2003). The eye-position data were obtained with a resolution of 0.7 at a sampling frequency of 30 Hz, and stored on digital tape for detailed off-line analysis. In the visual reaction-time task, a trial was started when the monkey pressed the lever. The monkey was required to remain pressing the lever throughout the trial. After the lever had been pressed, a fixation point (FP; 0.2 in diameter, 35 cd/m2) was presented against a uniform background (10 cd/m2) at the straight-ahead position of the eye being assessed. When the monkey had fixated the spot within a 2.0 or 2.5 electronic window for 0.5e5.5 s, a target stimulus was presented for 1.0 s at 1 of 54 possible peripheral positions. The duration of the fixation period was varied from trial to trial by means of a pseudorandom function, so that the monkey could not forecast the appearance of the target stimulus. We adopted Goldmann size III (4 mm2: w0.43 ) as the target-stimulus size. In the trials, the target stimulus was presented at 1 of 54 possible positions set according to the Central 24-2 program of the Humphrey Field Analyzer (HFA). The luminance of the target stimulus, which was added to the background luminance, is expressed on the decibel (dB) scale, as in the HFA. However, the luminance was limited to a range from 39 dB (0.40 cd/m2) to 10 dB (318 cd/m2) because of the limitations of the CRT monitor. If the monkey released its hand from the lever within 1.0 s of target-onset, we considered that the monkey had detected the target, and a drop of juice (0.5 ml) was administered as a reward. Then, a 1.0-s intertrial interval (ITI) began. If the monkey broke fixation at any time during the trial (i.e., before lever-releasing) or if it released the lever before the presentation of the target stimulus, the FP was turned off, the trial was aborted without a reward, and the ITI began following a punishment period of up to 5.0 s. We adopted the up-and-down method to plot the visual fields. Using this up-and-down method, we could continuously

measure the luminance-contrast threshold for the target stimulus. In the present study, the threshold value for each position was calculated as the mean luminance of the target stimulus from trials performed within 1 day (left control eye) or 2 days (right glaucomatous eye) using the up-and-down method. 2.4. Measurement of eye parameters We performed several ocular-testing sessions weekly (including IOP measurement, fundus photography, refractometry, and corneal opacity measurement) under intramuscular ketamine (10 mg/kg). IOP was measured in both eyes of each animal using a calibrated applanation pneumatonometer (PneumatonographÔ; Alcon Inc., TX, USA) with 0.4% oxibuprocaine hydrochloride (BenoxilÒ 0.4% solution, Santen Pharmaceutical Co. Ltd., Osaka, Japan) as local anesthetic. Fundus photographs were obtained using an ocular fundus camera (Genesis; Kowa Co. Ltd., Aichi, Japan). Refraction and corneal curvature radius were measured for each eye using an Auto Ref/Keratometer (ARK-700A; Nidek Co. Ltd., Aichi, Japan), and their values were used in the determination of the contact lens power needed to correct the reflection. 2.5. Perfusion and tissue enucleation After the final visual field test (16 weeks after the first laser application), the monkeys were perfused via the common carotid artery with 1 L of 0.9% saline containing 10 U/ml heparin at room temperature, followed by 1 L of 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS; pH 7.4). This was done under deep general anesthesia (produced by an intravenous injection of pentobarbital sodium), and eyes and brains were enucleated after the perfusion. 2.6. Histological examination of retina The retinal tissue processing for the histological analysis has been described previously (Shimazawa et al., 2006). Posterior portions of eye were washed three times with PBS (pH 7.4) and cut down to an area of central retina together with choroid and sclera (approximately 10 mm square and centered on the fovea). Each sample was embedded in paraffin, then retinal cross sections were cut vertically at 100 mm intervals (each section 3 mm thick) and stained with hematoxylin and eosin. Tissue samples for a maximum of 16 specific perimetry test sites were taken from comparable retinal locations in control and glaucomatous eyes as reported previously (Harwerth et al., 1999). In total, only 35 positions from the four eyes of the two monkeys were analyzed because of the technical difficulty of sample preparation for all locations. We adopted a constant conversion ratio of 1 mm retina per 5 of visual angle to determine the retinal locations (Perry and Cowey, 1985; Wassle et al., 1990). This ratio was convenient for our experiments because the value obtained for the distance from the fovea to the center of the optic nerve head per degree from the fixation point to

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the center of blind spot was consistent with the conversion ratio. Retinal damage was evaluated as follows, three sections from each eye being used for the morphometric analysis. Light-microscope images were photographed, and the cell counts in the ganglion cell layer (GCL) at a distance of 0.25 mm were measured on the photographs in a masked fashion by a single observer (M. Ookuma; see Acknowledgements). Data from maximum five samples were averaged for each position and used to evaluate the cell count in the GCL. 2.7. Histological examination of lateral geniculate nucleus

(Ylem, Rome, Italy). They were then washed with 0.01 M PBS and incubated with biotinylated anti-mouse IgG before being incubated with the avidinebiotineperoxidase complex for 30 min at room temperature, and finally developed using DAB peroxidase substrate. For quantification of Nissl-stained sections, the area of the LGN was measured in all six layers. Cell numbers in the Mand P-layers were estimated by counting (under a microscope fitted with a 40 objective) all neurons displaying clear nucleoli that were located within a sample volume of 250  188  5 mm (section thickness). Cell counts were derived from three regions per LGN sample section. 3. Results

Enucleated brains were cut into several sections, and these were postfixed by immersion in 4% paraformaldehyde in PBS (pH 7.4) for at least 48 h at 4  C. Each sample was embedded in paraffin, and coronal cross sections containing the lateral geniculate nucleus (LGN) were cut at 300 mm intervals (each section 5 mm thick) then stained with Nissl. For immunohistochemistry, coronal sections containing LGN were placed on slides and washed with 0.01 M PBS, then treated with 0.3% hydrogen peroxidase in methanol for 30 min at room temperature. Next, they were preincubated with 10% normal goat serum (Vector) in 0.01 M PBS for 30 min, then incubated for one day at 4  C with mouse antiglial fibrillary acidic protein (GFAP) monoclonal antibody

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3.1. Changes in intraocular pressure and visual fields in experimental glaucoma The intraocular pressure (IOP) of the monkeys was elevated and remained above baseline over the 15 weeks after the first laser-photocoagulation treatment (Fig. 1A,B). Although IOP was evaluated in each monkey, the degree of elevation differed between them, particularly in the second half of the post-treatment period. The period for which IOP was sustained at over 40 mmHg was weeks 1e11 and weeks 3e15 after the first laser treatment in monkeys A and B, respectively, while the maximum IOP value was 60.3 and 58.7 in monkeys A and B, respectively. Although quite slight corneal opacity was

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Fig. 1. Changes in intraocular pressure (IOP) and mean threshold of visual field after laser photocoagulation treatment in monkey A (A, C) and monkey B (B, D). IOP and visual field were monitored twice a week (except for a short period after the laser application). In the laser-treated right eyes (closed circle) IOP was elevated after the second laser application, and it remained above baseline for the remainder of the observation period. The mean threshold in the laser-treated eyes (closed circle) decreased gradually after the laser application. There were no significant changes in the left control eyes in either IOP or mean threshold (open circle). At each time-point, IOP was taken as the average of 3 measurements (means only). The mean threshold at each time-point was calculated from 52 measured thresholds, using the Central 24-2 program of the HFA, but excluding two points close to the blind spot (15 temporal and 3 above or below the horizontal meridian: mean  S.D.).

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Fig. 2. Visual field changes before and after laser photocoagulation treatment in monkey A (A) and monkey B (B). The threshold at each measurement-point was calculated from trials performed within 1 day (left control eye) or 2 days (laser-treated right eye). Each threshold between 10 and 19 dB is indicated in red, while those between 20 and 25 dB are indicated in blue. The values for mean threshold indicated in the lower part of each visual field were each calculated from 52 measured thresholds, but excluding two points close to the blind spot (boxed data, 15 temporal and 3 above or below the horizontal meridian). The data marked by asterisks (upper-right of each threshold value) were used for the comparison analysis between visual field sensitivity and number of retinal ganglion cells (see Fig. 4).

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observed temporally after the second laser application in both monkeys by the acute IOP elevation, these effects on visual field thought to be very small because both the fixation and motivation during the visual field measurement were not changed. Mean threshold changes in each monkey are illustrated in Fig. 1C and D. Mean threshold decreased rapidly after laser application in both monkeys, and reached a plateau about 7e8 weeks after the first laser application. The values obtained for the final threshold difference versus mean baseline threshold in each monkey were 8.99 dB (monkey A) and 9.63 dB (monkey B). In contrast, in control eyes the corresponding values were 1.44 dB in monkey A and 0.35 dB in monkey B. In monkey A, the threshold in the nasal field was decreased by over 5 dB at most points (versus the baseline threshold values) (Fig. 2A). The threshold changes versus baseline in the temporal-central field were less than 3 dB. The mean threshold values obtained for the nasal half-field (total 26 points: the 2 most peripheral points being omitted), temporal half-field (total 24 points: 2 points near the blind spot being omitted), superior half-field (total 26 points: 1 point near the blind spot being omitted), and inferior half-field (total 26 points: 1 point near the blind spot being omitted) were 18.2  6.2 dB, 22.9  5.8 dB, 18.7  6.8 dB, and 21.4  6.2 dB, respectively. These mean threshold values indicate that the nasal half-field had the lowest sensitivity in monkey A. In monkey B, all measured thresholds at 15 weeks after the first laser application were decreased by 5 dB or more versus baseline (Fig. 2B). Central points and

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a few points near the central field showed comparatively mild defects. The mean threshold values for the nasal half-field, temporal half-field, superior half-field, and inferior half-field were 19.6  4.7 dB, 19.8  4.8 dB, 17.2  5.2 dB, and 21.5  3.7 dB, respectively, indicating that the superior halffield had the lowest sensitivity in monkey B. There was no corneal opacity at the final visual field test (15 weeks after the first laser application). 3.2. Changes in fundus and histological sections in experimental glaucoma The upper two rows of photographs in Fig. 3 are fundus photographs taken before and after the laser application in each monkey. The fundus was not changed in the left control eyes after laser application to the right eyes, but changes were apparent in the laser-treated right eye in both monkeys. Deeper cup depth was observed in both monkeys. Thinner rim area was also observed, especially in inferior side than superior side of optic nerve head (ONH) in both monkeys. These trends were reasonable because inferior side of ONH corresponds to the position of superior in visual field that showed lower sensitivity than inferior visual field (Fig. 2). The histological changes in the ONH after the final visual field evaluation are shown for each monkey in the lowest row of photographs in Fig. 3. Apparent changes in the thickness of the nerve fiber layer were observed in the right eye of each monkey (versus the contralateral left control eye).

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Fig. 3. Fundus photographs and optic nerve head cross-sections in monkey A (A) and monkey B (B). Laser photocoagulation treatment was performed in the right eye. Photographs shown in upper panels and middle panels in both (A) and (B) are fundus photographs taken, respectively, before and after laser application. Lower panels show optic nerve head cross-section in each monkey 16 weeks after the first laser application.

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RGC density (Log10(number/mm)) Fig. 4. Comparison between number of retinal ganglion cells (RGC) and visual field sensitivity in monkeys. The relationship was analyzed for three eccentricities: A (3 , 3 ); B (9 , 9 ); and C (15 , 15 ). The maximum number of RGC decreased as the eccentricity from the fovea increased. The visual field sensitivity values indicate the threshold at each measurement-point at the last visual field measurement (15 weeks after the first laser application), the relevant positions being marked by asterisks in Fig. 2.

The relationship between the last visual field sensitivity measurement and the density of retinal ganglion cells (RGC) was analyzed at three points (3 , 3 ), (9 , 9 ), and (15 , 15 ) in each quadrant of the visual field of both eyes (Fig. 4). The visual field sensitivity and RGC density showed a significant correlation at each point (Fig. 4; Spearman correlation test; A: r ¼ 0.6686, p ¼ 0.0046; B: r ¼ 0.7882, p ¼ 0.0040; C: r ¼ 0.7619, p ¼ 0.0280). The maximum density of RGC in the untreated eye decreased with the increment in the eccentricity from the fovea (open circles in Fig. 4); viz. about 600 at (3 , 3 ), 120 at (9 , 9 ), and 60 at (15 , 15 ). Although the RGC density and visual field sensitivity decreased in the

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laser-treated eye, the correlation between RGC density and visual field sensitivity differed with the degree of eccentricity, the slope increasing as the eccentricity increased. The slope values were 8.4, 13.0, and 35.4 at (3 , 3 ), (9 , 9 ), and (15 , 15 ), respectively. These results suggest that greater

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acute changes (larger curvature) occurred at greater eccentricity increments (Fig. 5A). On the other hand, Y-intercept values were 9.3, 3.1, and 30.0 at (3 , 3 ), (9 , 9 ), and (15 , 15 ), respectively. These results suggest that the Y-intercept function tended to decrease with eccentricity increments (Fig. 5B).

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Fig. 6. Photograph of Nissl-stained coronal section through the left lateral geniculate nucleus (A), and comparison between right (ipsilateral to the high IOP eye) and left (contralateral to the high IOP eye) nuclei in the Nissl-stained sections (B). Boxed areas in (A) are shown at higher magnification in (B) [numbered as in (A)]. Scale bars: 100 mm (A), 30 mm (B).

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3.4. Histological and glial fibrillary acidic protein expression changes in lateral geniculate nucleus The neurons in the lateral geniculate nucleus (LGN) displayed different patterns of change on the ipsilateral (right) and contralateral (left) sides. On the ipsilateral side, the retinal input from the high IOP eye projects to layer 2 (M-layer) and to layers 3 and 5 (P-layers), and obvious neuronal losses were observed in these layers (versus the contralateral untreated eye) (Fig. 6B). On the contralateral side, the retinal input from the high IOP eye projects to layer 1 (M-layer) and to layers 4 and 6 (P-layers), and as predicted, neuronal losses were confirmed in these layers (Fig. 6B). On the whole, there was a decrease in the number of neurons in each LGN layer in three sections (Fig. 6A, Table 1), although the percentage reduction was greater in the P-layers (about 15e25%) than in the M-layer (about 5e15%). Glial fibrillary acidic protein (GFAP) expression was confirmed immunohistochemically, all LGN layers being stained (Fig. 7A). The intensity of GFAP staining (brown) was upregulated in the layers that receive a retinal input from the high IOP eye (ipsilateral layers: 2, 3, 5; contralateral layers: 1, 4, 6) than in those receiving from the contralateral untreated eye (Fig. 7B). 4. Discussion In the present study, we examined the changes in the visual field, RGC density in the retina, and histology and GFAP expression in LGN in high IOP monkeys. Visual field changes were first apparent at 2e3 weeks and reached a plateau at about 7e8 weeks after the first laser application. We previously reported that morphometric changes in the retina started fairly rapidly after laser application (Shimazawa et al., 2006) and reached plateau about 5 weeks after Table 1 Cell numbers in each layer of LGN Layer

1 2 3 4 5 6

Mean (A, B) Ipsi

Contra

44.6 (51.4, 37.9) 39.1 (42.8, 35.4) 44.6 (47.8, 41.3) 60.6 (68, 53.1) 38.1 (34.3, 31.9) 55 (58.2, 51.8)

37.3 (38.5, 36) 42.4 (44.3, 40.5) 56.5 (59.6, 53.4) 50.3 (59, 41.6) 49.7 (45.6, 53.8) 41.7 (37.8, 45.6)

Density (mm2)

% of Ipsilateral

Ipsi

Contra

Ipsi

951.4

795.8

100

84

834.1

904.5

100

108

951.5

1205.3

100

127

1292.8

1073.1

100

83

812.8

1060.3

100

130

1173.3

889.6

100

76

Contra

Cell number in each layer was estimated by counting (under a microscope with a 40 objective) all neurons displaying clear nucleoli located within a sample volume of 250  188  5 mm (section thickness). ‘‘Mean’’ cell counts were derived from three regions per LGN sample section (numbers within parentheses represent the counts for the individual monkeys), and then ‘‘density’’ and ‘‘% of ipsilateral’’ values were calculated.

the first laser application. IOP of our two monkeys were reached between 50 and 60 mmHg more than 5 weeks after the second laser application. Although we performed IOP measurements twice a week and also did not observe any ischemic fundus changes among the experimental term, we could not completely deny that IOP spiking between the measurements had occurred and resulted in transient retinal ischemia. In rat, IOP over 60 mmHg have affected several retinal components including RGC (Bui et al., 2005). On the other hand, there were few monkeys that did not show visual field losses even if these IOP reached near 60 mmHg in a previous report (Harwerth et al., 1997). Thus, we think both mechanical and ischemic stresses are included in the early visual field changes in our monkey, and we also think that it is difficult to determine the threshold (critical) IOP level of functional loss caused by mechanical or ischemic stress. In the analysis of the relationship between the visual sensitivity to the ganglion cell density using logelog coordinates, the linear regression of the visual field sensitivity to the normal RGC density was decreased by the eccentricity increment, and this analysis also showed the trends that slope was increasing and Y-intercept was decreasing with the eccentricity increment. Although our results were obtained from only two monkeys, quite similar trends were confirmed in the previous report using rhesus monkeys (Harwerth et al., 2004). Thus, we suspect that these trends were consistent within these species, and these results suggest that the rate of one RGC contribution to visual sensitivity was increased by the degree of eccentricity. These differences of one RGC contribution to sensitivity among the eccentricity may be one reason for specific visual field changes to any injuries including glaucoma. In previous reports, although the total LGN neuron number and the mean soma size were both decreased, cell density was slightly increased in high IOP monkeys compared with na€ıve ones (Weber et al., 2000; Yucel et al., 2000, 2001, 2003). We could not perform such analyses, because we did not employ na€ıve monkeys in our study. However, one report has compared ‘‘affected’’ with ‘‘unaffected’’ layers in the same high IOP monkeys (Weber et al., 2000). To judge from their figures, the ‘‘affected’’ layer had a huge soma-size shrinkage and a slight trend towards a cell-density decrement (compared to the ‘‘unaffected’’ layers) in a monkey that had experienced over 8 weeks IOP elevation, and the changes were more profound in M-layers than in P-layers. Although in our study both M-layers and P-layers showed decrements in LGN neuron density, the decrement was slightly greater in P-layers and similar findings that neuronal change was not selective for M-layers have also been observed in other studies (Morgan et al., 2000; Vickers et al., 1997; Yucel et al., 2000). On the other hand, only few studies have reported on neuronal loss of LGN in human glaucoma (Chaturvedi et al., 1993; Gupta et al., 2006). Although one study suggested neuronal loss was occurred only in M-layers even in end-stage of glaucoma (Chaturvedi et al., 1993), another group reported M-layers were more affected than P-layers that also had a neuronal loss in the middle stage of glaucoma (MD ¼ 7.38 dB) (Gupta et al., 2006). There is thus some discrepancy in the affected

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779

A 6 5 4 3 2 1

Right

Left

Right

Left

B

1

4

2

5

3

6

Fig. 7. Photograph of GFAP-immunostained coronal section through the left lateral geniculate nucleus (A), and comparison between right (ipsilateral to the high IOP eye) and left (contralateral to the high IOP eye) nuclei in the GFAP-immunostained sections (B). Boxed areas in (A) are shown at higher magnification in (B) [numbered as in (A)]. Scale bars: 100 mm (A), 30 mm (B).

layers and amounts in LGN between the studies, but it may be that some neuronal reconstitution occurs in both layers of LGN when obvious visual field changes occur in monkeys and humans. Although there are many reports of GFAP expression in the retina, its expression in LGN is not fully understood.

Reportedly, GFAP expression in LGN is upregulated when either the neuronal input from the retina (Canady et al., 1994; Gonzalez et al., 2006) or feedback transduction from the visual cortex (Agarwala and Kalil, 1998) is completely abolished. In the present study, we found that high IOP induced a partial visual field loss (about a 9 dB reduction in

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M. Sasaoka et al. / Experimental Eye Research 86 (2008) 770e782

mean threshold versus baseline) and also increased GFAP expression in those LGN layers that receive a neuronal input from the high IOP eye (versus those receiving from the contralateral untreated one). This phenomenon is interesting because an increase in GFAP expression is usually observed in neuronal tissue that has been undergoing neuronal reorganization after a lesion (Mauch et al., 2001; Pfrieger and Barres, 1997; Ullian et al., 2001). Thus, our results suggest that some LGN neuronal degeneration and/or reconstitution may occur in glaucoma patients even if visual field damage is only moderate. Although upregulation of GFAP has been reported to be a hallmark of neuronal injury (O’Callaghan and Sriram, 2005), the precise function of GFAP has not been determined. To judge from some studies using GFAP-knockout mice, GFAP may (a) serve to maintain both normal processes in astrocytes and the bloodebrain barrier (Liedtke et al., 1996), and (b) play a neuroprotective role in several injuries, such as traumatic brain injury (Nawashiro et al., 1998), cerebral ischemia (Nawashiro et al., 2000), and excitotoxic injury (Otani et al., 2006). Moreover, upregulation of GFAP is essential for the retention of GLAST (one of the glutamate transporters in astrocytes) after a hypoxic insult (Sullivan et al., 2007). Since downregulation of GLAST has been observed in the aged brain (Nickell et al., 2007), in the brain at early clinical stages of Alzheimer’s disease (Jacob et al., 2007), and in the retina of glaucomatous patients (Naskar et al., 2000), GFAP may play a protective role against these degenerative phenomena. Thus, high IOP-induced neuronal degeneration in LGN may induce GFAP upregulation, and GFAP may play a neuroprotective role against the effects of several injuries, including excess glutamate excitotoxicity. If this is true, effective protection against neurodegenerative processes in glaucoma may require not only prevention of RGC death by focal treatment application, but also wide protective treatment directed towards the LGN and visual cortex (by systemic administration). By our use of a personal computer-based perimetry system, we could detect visual field changes in the laser-induced highIOP monkey. To judge from our analysis of RGC density and visual field sensitivity, the rate of one RGC contribution to visual sensitivity was increased by retinal eccentricity. Furthermore, our histological and immunohistochemical analysis of LGN suggests that neuronal degeneration may occur even if the visual field changes are only moderate, and this neuronal degeneration may also induce changes in glial function within the CNS. Although further studies will be needed using a greater number of monkeys, our observations should help to elucidate the pathophysiology of glaucoma. Acknowledgments We thank Dr. K. Matsuda (Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology), Mr. S. Nagumo (Primate Research Institute, Kyoto University), Dr. K. Kuraoka (National Institute of Neuroscience, National Center of Neurology and Psychiatry),

Dr. T. Taniguchi and Ms. M. Ookuma (Santen Pharmaceutical Co. Ltd.) for their technical assistance, and Dr. G. Tomita (University of Tokyo School of Medicine) for his help. We are deeply grateful to Professors S. Kojima (Keio University), C. Matsumoto (Kinki University School of Medicine) and R.S. Harwerth (College of Optometry, University of Houston) for their support and guidance. This work was supported by the Cooperation Research Program between Kyoto University and Santen Pharmaceutical Co. Ltd. (2000–2002) and in part by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sport, Science and Technology, Japan (No. 19592031, No. 18209053). References Agarwala, S., Kalil, R.E., 1998. Axotomy-induced neuronal death and reactive astrogliosis in the lateral geniculate nucleus following a lesion of the visual cortex in the rat. J. Comp. Neurol. 392, 252e263. Bowd, C., Zangwill, L.M., Berry, C.C., Blumenthal, E.Z., Vasile, C., SanchezGaleana, C., Bosworth, C.F., Sample, P.A., Weinreb, R.N., 2001. Detecting early glaucoma by assessment of retinal nerve fiber layer thickness and visual function. Invest. Ophthalmol. Vis. Sci. 42, 1993e2003. Bui, B.V., Edmunds, B., Cioffi, G.A., Fortune, B., 2005. The gradient of retinal functional changes during acute intraocular pressure elevation. Invest. Ophthalmol. Vis. Sci. 46, 202e213. Canady, K.S., Olavarria, J.F., Rubel, E.W., 1994. Reduced retinal activity increases GFAP immunoreactivity in rat lateral geniculate nucleus. Brain Res. 663, 206e214. Chaturvedi, N., Hedley-Whyte, E.T., Dreyer, E.B., 1993. Lateral geniculate nucleus in glaucoma. Am. J. Ophthalmol. 116, 182e188. Crawford, M.L., Harwerth, R.S., Smith 3rd, E.L., Shen, F., Carter-Dawson, L., 2000. Glaucoma in primates: cytochrome oxidase reactivity in parvo- and magnocellular pathways. Invest. Ophthalmol. Vis. Sci. 41, 1791e1802. Crawford, M.L., Harwerth, R.S., Smith 3rd, E.L., Mills, S., Ewing, B., 2001. Experimental glaucoma in primates: changes in cytochrome oxidase blobs in V1 cortex. Invest. Ophthalmol. Vis. Sci. 42, 358e364. Dreyer, E.B., Zurakowski, D., Schumer, R.A., Podos, S.M., Lipton, S.A., 1996. Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma. Arch. Ophthalmol. 114, 299e305. Eng, L.F., Ghirnikar, R.S., Lee, Y.L., 2000. Glial fibrillary acidic protein: GFAP-thirty-one years (1969e2000. Neurochem. Res. 25, 1439e1451. Finch, C.E., 2003. Neurons, glia, and plasticity in normal brain aging. Neurobiol. Aging 24 (Suppl. 1), S123eS127. discussion S131. Flammer, J., Orgul, S., Costa, V.P., Orzalesi, N., Krieglstein, G.K., Serra, L.M., Renard, J.P., Stefansson, E., 2002. The impact of ocular blood flow in glaucoma. Prog. Retin. Eye Res. 21, 359e393. Frishman, L.J., Shen, F.F., Du, L., Robson, J.G., Harwerth, R.S., Smith 3rd, E.L., Carter-Dawson, L., Crawford, M.L., 1996. The scotopic electroretinogram of macaque after retinal ganglion cell loss from experimental glaucoma. Invest. Ophthalmol. Vis. Sci. 37, 125e141. Frishman, L.J., Saszik, S., Harwerth, R.S., Viswanathan, S., Li, Y., Smith 3rd, E.L., Robson, J.G., Barnes, G., 2000. Effects of experimental glaucoma in macaques on the multifocal ERG. Multifocal ERG in laserinduced glaucoma. Doc. Ophthalmol. 100, 231e251. Gomi, H., Yokoyama, T., Fujimoto, K., Ikeda, T., Katoh, A., Itoh, T., Itohara, S., 1995. Mice devoid of the glial fibrillary acidic protein develop normally and are susceptible to scrapie prions. Neuron 14, 29e41. Gonzalez, D., Satriotomo, I., Miki, T., Lee, K.Y., Yokoyama, T., Touge, T., Matsumoto, Y., Li, H.P., Kuriyama, S., Takeuchi, Y., 2006. Changes of parvalbumin immunoreactive neurons and GFAP immunoreactive astrocytes in the rat lateral geniculate nucleus following monocular enucleation. Neurosci. Lett. 395, 149e154. Gordon, M.O., Beiser, J.A., Brandt, J.D., Heuer, D.K., Higginbotham, E.J., Johnson, C.A., Keltner, J.L., Miller, J.P., Parrish 2nd, R.K., Wilson, M.R., Kass, M.A., 2002. The Ocular Hypertension Treatment

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