Effect of glaucoma on the retinal glutamate/glutamine cycle activity

©2005 FASEB The FASEB Journal express article 10.1096/fj.04-3313fje. Published online May 2, 2005. Effect of glaucoma on the retinal glutamate/glutamine cycle activity María Cecilia Moreno,* Pablo Sande,* Hernán Aldana Marcos,† Nuria de Zavalía,* María Inés Keller Sarmiento,* and Ruth E. Rosenstein* *Laboratorio de Neuroquímica Retiniana y Oftalmología Experimental, Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, CONICET, Buenos Aires; and †Laboratorio de Histología, Facultad de Medicina, Universidad de Morón, Pcia. de Buenos Aires, Argentina

Corresponding author: Ruth E. Rosenstein, Departamento de Bioquímica Humana, Facultad de Medicina, UBA Paraguay 2155, 5º P, (1121), Buenos Aires, Argentina. E-mail: [email protected] ABSTRACT

Glutamate-induced excitotoxicity has been proposed to mediate the death of retinal ganglion cells in glaucoma. The metabolic dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. Thus, the aim of the present work was to study the retinal glutamate/glutamine activity in eyes with hypertension induced by intracameral injections of hyaluronic acid (HA). For this purpose, weekly injections of HA were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with saline solution. At 3 or 10 weeks of treatment, glutamate and glutamine uptake and release were assessed using [3H]-glutamate and [3H]-glutamine as radioligands, respectively. In addition, glutamine synthetase activity was assessed by a spectrophotometric assay, whereas glutaminase activity was measured through the conversion of [3H]-glutamine to [3H]-glutamate. At 3 weeks of treatment with HA, a significant decrease (P<0.01) in glutamate uptake and glutamine synthetase activity was observed. Glutamine uptake and release, as well as glutaminase activity, were significantly increased (P<0.01) in eyes injected with HA for 3 weeks compared with vehicle-injected eyes, whereas [3H]-glutamate release did not change in hypertensive eyes. Only the changes in glutamine synthetase activity persisted at 10 weeks of treatment with HA. These results indicate a significant alteration in the retinal glutamate/glutamine cycle activity in hypertensive eyes. Since these changes preceded both functional and histological alterations induced by ocular hypertension, these results support the involvement of glutamate in glaucomatous neuropathy.

Key words: excitotoxicity • glutamatergic neurons • hyaluronic acid

laucoma is a leading cause of blindness worldwide, characterized by specific visual field defects due to the loss of retinal ganglion cells and damage to the optic nerve head. The result is a patchy loss of vision generally in a peripheral to central manner. It is estimated G

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that half of those affected may not be aware of their condition because symptoms might not occur during the early stages of the disease. When vision loss appears, considerable permanent damage has already occurred. Medications and surgery can help slow the progression of some forms of the disease, but at present no cure is available.

Elevated intraocular pressure (IOP) is one of the most important risk factors for development of glaucoma. In fact, reduction of IOP by medical and/or surgical means continues to be the mainstay of therapy for glaucoma. The underlying mechanisms that link elevated IOP to glaucomatous ganglion cell death are not fully understood. An experimental model system of pressure-induced optic nerve damage would greatly facilitate the understanding of the cellular events leading to ganglion cell death and how these events are influenced by IOP and other risk factors. We have developed a new model of glaucoma in rats through intracameral injections of hyaluronic acid (HA). The acute or chronic injections of HA in the rat anterior chamber significantly increase IOP as compared with the vehicle-injected contralateral eye (1). Several groups have developed various ways to increase IOP in the rat eye, generally by impeding the outflow of aqueous humor (2–4). All of these models have both advantages and disadvantages. Although multiple injections of HA may be needed to obtain a sustained hypertension, we have shown that the injection procedure itself does not affect IOP. On the contrary, several advantages support the usefulness of our model: 1) a highly consistent hypertension may be achieved; 2) it might have a reasonably long course; 3) daily variations in IOP persist in HA-injected eyes; 4) in contrast to other models, in all likelihood, HA does not impede the outflow; and 5) it is easy to perform. Furthermore, since the HA-induced hypertension is significantly reduced by the local and acute application of hypotensive drugs, this model may also be utilized for pharmacological studies (1). More recently, we showed that the chronic administration of HA for 6–10 weeks, rather than 3 weeks, significantly decreases the scotopic electroretinographic activity (5, 6). After 10 weeks of treatment, a significant loss of ganglion cell layer cells and optic nerve axons were detected in eyes that received HA, as compared with saline injected-eyes (5, 6). Based on both functional and histological evidences, these results indicate that intracameral injections of HA appear to mimic central features of primary open-angle glaucoma, and therefore it may be a useful tool to understand this ocular disease.

Although increased IOP is probably the most important risk factor in primary-angle open glaucoma, several concomitant factors, like elevation of glutamate levels, disorganized nitric oxide (NO) metabolism, and oxidative damage, among others, could significantly contribute to the neurodegeneration (for review, see 7). Recently, we demonstrated a significant decrease of the retinal antioxidant defense system activity in hypertensive eyes (8).

Glutamate is the main excitatory neurotransmitter in the retina, but it is toxic when present in excessive amounts. Retinal tissue is in fact an established paradigm for glutamate neurotoxicity for several reasons: (i) insult leads to accumulation of relatively high levels of glutamate in the extracellular fluid (9); (ii) administration of glutamate leads to neuronal cell death (10); and (iii) glutamate receptor antagonists can protect against neuronal degeneration (11). Thus, an appropriate clearance of synaptic glutamate is required for the normal function of retinal excitatory synapses and for the prevention of neurotoxicity. Glial cells, mainly astrocytes and Müller glia, surround glutamatergic synapses and express glutamate transporters and the glutamate-metabolizing enzyme glutamine synthetase (12, 13). Glutamate is transported into glial cells and amidated by glutamine synthetase to the non-toxic aminoacid glutamine.


Glutamine is then released by the glial cells and taken up by neurons, where it is hydrolyzed by glutaminase to form glutamate again, completing the retinal glutamate/glutamine cycle (14, 15). In this way, the neurotransmitter pool is replenished and glutamate neurotoxicity is prevented.

Glutamatergic injury has been proposed to contribute to the death of retinal ganglion cells in glaucoma. This hypothesis is supported by the demonstration that vitreal glutamate is elevated in glaucomatous dogs (16) and quail with congenital glaucoma (17). In addition, high glutamine levels have been found in retinal Müller cells of glaucomatous rat eyes (18). In contrast, other authors showed no significant elevation of glutamate in the vitreous of patients with glaucoma (19), or in rats (20) and monkeys with anatomic and functional damage from experimental glaucoma (21, 22). In any case, it seems limited to assume that high levels of glutamate in the vitreous are a necessary condition for excitotoxicity to be involved in glaucomatous neuropathy. The local concentration of glutamate at the membrane receptors of ganglion cells is the important issue for toxicity. This could be very different from the level in samples of vitreous. Vitreous humor must be removed for experimental measurement by a process that inevitably disturbs its state before removal. These manipulations could themselves alter the measured amount of glutamate. At present, no tools can directly assess retinal glutamate synaptic concentrations in vivo. The aim of the present work was to analyze the retinal mechanisms that regulate glutamate clearance and recycling in eyes with high IOP induced by intracameral injections of HA.

MATERIALS AND METHODS

Animals and tissues

Male Wistar rats (average weight, 200±40 g) were housed in a standard animal room with food and water ad libitum under controlled conditions of humidity and temperature (21±2°C). The room was lighted by fluorescent lights that were turned on and off automatically every 12 h (on from 6.00 AM to 6.00 PM). Rats were anesthetized with ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (0.5 mg/kg) administered intraperitoneally. With a syringe (Hamilton, Reno, NV) and a 30-gauge needle, 25 µl of HA (10 mg/ml in saline solution; catalog no. H1751; Sigma Chemical Co., St. Louis, MO) were injected into one eye of anesthetized rats, and an equal volume of vehicle (saline solution) was injected in the fellow (control) eye as previously described (1). Briefly, the eyes were focused under a Colden surgical microscope (Briuolo, Buenos Aires, Argentina) with coaxial light. The needle moved through the corneoscleral limbus to the anterior chamber with the bevel down. When the tip of the bevel reached the anterior chamber, the liquid progressively increased the chamber’s depth, separating the needle from the iris, and avoiding needle-lens contact. Injections were applied at the corneoscleral limbus beginning at hour 12 and changing the site of the next injection hourly, by rotating the head to achieve better access to the limbus. The injections and IOP assessments were performed after applying 1 drop of 0.5% proparacaine hydrochloride to each eye. Of 154 rats, 10 showing cataract and 2 with phthisis bulbi were excluded from the experiments. In addition, a group of 20 rats that was handled and anesthetized once a week for 3 weeks, but not injected in either eye, was included in this study (control group). After IOP assessment, animals were sacrificed by decapitation. Eyeballs were quickly enucleated after death, and the corneas were removed. The lens and vitreous were dissected under surgical microscope, and the retinas were detached by blunt dissection. Before homogenization, retinas were examined to eliminate possible choroidal tissues and were processed as described below for each protocol. To gain insight into the


development of the pathological mechanisms leading to ganglion cell death, we used retinas from rats at 3 and 10 weeks of treatment with HA or vehicle. These time points were chosen to compare a time point with no signs of retinal disease (3 weeks) with one in which both functional and histological changes were evident (10 weeks).

All animal use procedures were in strict accordance with the NIH Guide for Care and Use of Laboratory Animals.

IOP assessment

A tonometer (TonoPen XL; Mentor, Norwell, MA) was used to assess IOP in conscious, unsedated rats, as described (23). All IOP determinations were assessed by operators who were blind to the treatment applied to each eye. Animals were wrapped in a small towel and held gently, with one operator holding the animal and another making the readings. Five IOP readings were obtained from each eye by using firm contact with the cornea and omitting readings obtained as the instrument was removed from the eye. The mean of these readings was recorded as the IOP for that eye on that day. Mean IOPs from each rat were averaged, and the resultant mean IOP was used to compute the group mean IOP ± SE. IOP was assessed weekly, before the new injection. IOP measurements were performed at the same time each week (between 11:00 and 12:00 h) to correct for diurnal variations in IOP.

Light microscopy

At 3 or 10 weeks of treatment with HA or vehicle, 5 eyes/group were analyzed by light microscopy. Eyes were enucleated after anesthetic overdose and immersed immediately in a fixative containing 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 1 h. The nictitans membrane was maintained in each eye to facilitate orientation. The cornea and the lens were carefully removed, and the posterior portions were fixed for an additional 12 h-period in the same fixative. Eyecups were then dehydrated, embedded in paraffin, sectioned with a microtome at 2 µm thickness, and stained with hematoxylin and eosin. Each section was cut along the horizontal meridian of the eye through the optic nerve head, perpendicular to the retinal surface. Microscopic images were digitally captured with a Nikon Eclipse E400 microscope (illumination: 6-V halogen lamp, 20W, equipped with a stabilized light source) via a Sony SSC-DC50 camera. The microscope was set up properly for Koehler illumination. The camera output was digitized into a 520 × 390 pixel matrix (each pixel with 0–255 grey levels) with a Leadteck® WinView 601 video capture card, displayed on a computer monitor, and saved as an image of 24 bit RGB in BMP format. The digitalized images were transferred to a Scion Image for Windows analysis system (Scion Corporation Beta 4.0.2). Retinal morphometry was evaluated as described by Takahata et al. (24) with minor modifications. Three sections were randomly selected from each eye. Nine microscopic images at 1 mm from the temporal edge of the optic disc were digitally analyzed. The light microscope was adjusted to level 4 and a 40 × CF E achromat objective was used. At the magnification utilized, each pixel of the image corresponds to 0.31 µm, and each field in the monitor represented a tissue area of 19318.7 µm2. The thickness (in µm) of the inner plexiform layer (IPL), inner nuclear layer (INL), outer nuclear layer (ONL), and total retina was measured. The number of cells in the ganglion cell layer (GCL) was calculated by linear cell density (cells per 100 µm). For each eye, results obtained from three separate sections were averaged and the mean


of five eyes was recorded as the representative value for each group. No attempt was made to distinguish cell types in the GCL for enumeration of cell number. Observers masked to the protocol used in each eye performed the morphometric analysis.

Fundus photography

At 3 or 10 weeks of treatment with HA or vehicle, 5 rats/group were anesthetized as described before, pupils were dilated with 2.5% phenylephrine, and 0.5% proparacaine (Alcon Laboratories, Argentina) was applied for topical anesthesia. Following complete dilation, the anesthetized animal was placed in lateral recumbency under the Colden microscope with coaxial light and positioned with one holding hand. The rat fundus was visualized with the application of a slide glass, with a drop of 2.5% methylcellulose (Poen Laboratories, Argentina) placed on the contact area of the cornea. One person adjusted the animal’s position until the optic disc came into view for the person who is viewing trough a coaxial microscope with a digital camera adapted. A holder was used for alignment, adjusting light reflexes away from the disc by slight turning and tilting of the cover slip, and maintaining focus by avoiding the minimum movement of the anesthetized animals. When satisfactory focus, centering, and light reflex location were obtained, the operator activated the camera. A digital camera (Sony, Cyber-shot, 3.2 mega pixels, Japan) adapted to a Colden coaxial microscope was used for the imaging.

L-[3H]-Glutamate and L-[3H]-glutamine uptake assessment

The influx of L-[3H]-glutamate and L-[3H]-glutamine was assessed in a crude synaptosomal fraction of rat retinas. Retinas were homogenized (1:9 w/v) in 0.32 M sucrose containing 1 mM MgCl2 and centrifuged at 900 × g for 10 min at 4° C. Nuclei-free homogenates were further centrifuged at 30,000 × g for 20 min. The pellet was immediately resuspended in buffer HEPES-Tris, containing 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, (adjusted to pH 7.4 with Tris base) and aliquots (100–300 µg protein/100 µl) were incubated with 100 µl of L-[3H]-glutamate or L-[3H]-glutamine (500,000–800,000 dpm/tube, specific activity 17.25 Ci/mmol and 51 Ci/mmol, respectively). After 5 min, amino acids uptake was terminated by adding 4 ml of ice-cold HEPES-Tris buffer. The mixture was immediately poured onto Whatman GF/B filters under vacuum. The filters were washed twice with 4 ml-aliquots of ice-cold buffer, and the radioactivity on the filters was counted in a liquid scintillation counter. Non-specific uptake of L-[3H]-glutamate or L-[3H]-glutamine into synaptosomes was assessed by adding an excess of glutamate or glutamine (10 mM), respectively.

Glutamine synthetase assessment

Each retina was homogenized in 200 µl of 10 mM potassium phosphate, pH 7.2. Glutamine synthetase activity was assessed as described (25). Reaction mixtures contained 150 µl of retinal homogenates and 150 µl of a stock solution (100 mM imidazole-HCl buffer, 40 mM MgCl2, 50 mM β-mercaptoethanol, 20 mM ATP, 100 mM glutamate, and 200 mM hydroxylamine, adjusted to pH 7.2). Tubes were incubated for 15 min at 37°C. The reaction was stopped by adding 0.6 ml of ferric chloride reagent (0.37 M FeCl3, 0.67 M HCl, and 0.20 M trichloroacetic acid). Samples were placed for 5 min on ice. Precipitated proteins were removed by centrifugation, and the absorbance of the supernatants was read at 535 nm against a reagent blank. Under these conditions, 1 µmol of γ-glutamylhydroxamic acids gives an absorbance of 0.340. Glutamine


synthetase-specific activity was expressed as µmoles of γ-glutamylhydroxamate per hour per milligram of protein.

L-[3H]-Glutamate and L-[3H]-glutamine release

For release studies, crude synaptosomal fractions were incubated for 30 min at 37°C with [3H]-glutamate or L-[3H]-glutamine (1,000,000–1,500,000 dpm/retina) in 500 µl of HEPES-Tris buffer. Synaptosomal fractions were washed by centrifugation in fresh buffer in order to remove the excess of radioligand, and incubated for 10 min with gentle shaking in 500 µl of the same buffer or in a high K+ buffer (50 mM) in which osmolarity was conserved by equimolar reduction of Na+ concentration. Synaptosomes were centrifuged, pellets were digested with hyamine hydroxide, and radioactivity in the medium and that incorporated into the tissue was determined in a scintillation counter. Fractional release was calculated as the ratio: radioactivity released/total radioactivity uptake by the tissue. Greater than 80 and 85% of the released radioactivity was identified as authentic glutamate or glutamine, respectively, by thin-layer chromatography.

Glutaminase activity assessment

Glutaminase activity was assessed as described (26). Each retina was homogenized in 40 µl of 0.1% Triton X-100 in 7.5 mM Tris HCl, pH 8.8. The assay mixture contained 20 µl of retinal homogenate (200–400 µg of proteins), 50 mM glutamine, 0.2–0.5 µCi L-[3H]-glutamine, and 63 mM potassium phosphate, pH 8.2, in a total volume of 100 µl. Tubes were incubated for 1 h at 30°C, with gentle agitation. The reaction was stopped by adding 1 ml of cold 20 mM imidazole, pH 7.0. Samples were briefly centrifuged, and the supernatants were applied to 0.6 × 3.5 cm beds of anion exchange resin (Dowex, AG1-X2, 200–400 mesh hydroxide form, Bio-Rad Laboratories) previously charged with 1M HCl and washed with water. The reaction substrate was removed with 6 ml of imidazole buffer, which was discarded, and the reaction product was eluted with 3 ml of 0.1M HCl. Aliquots of this fraction were mixed with scintillation cocktail for measurement of radioactivity. Blanks were determined from samples lacking retinal homogenates. Glutaminase-specific activity was expressed as micromoles of glutamate per milligram protein per hour.

Protein content was determined by the method of Lowry et al. (27), using bovine serum albumin as the standard.

Statistical analysis of results was made by a Student's t-test or by a two-way analysis of variance (ANOVA) followed by a Dunnett's test, as stated.

RESULTS

Table 1 summarizes the average IOP of rats injected weekly with HA (in one eye) or vehicle in the other for 3 or 10 weeks. At these intervals, a significant increase of IOP (P<0.01) was observed in the eyes injected with HA as compared with the respective controls. No differences in the IOP of vehicle-injected eyes were detected between these time points or between non-injected and vehicle-injected eyes. The posterior segments of five eyes of rats were examined by light microscopy at 3 and 10 weeks of treatment with HA or vehicle. We found no differences


between control and eyes with elevated IOP in the outer retinal layers, as shown in Figure 1. However, we observed a significant loss of GCL cells in HA-injected eyes for 10 weeks. The mean number/100 µm ± SE of GCL cells at 3 weeks of treatment was 8.6 ± 0.8, and 9.0 ± 0.7 for vehicle and HA, respectively, while at 10 weeks of treatment it was for vehicle: 8.8 ± 0.9, and for HA: 5.0 ± 0.6 (P<0.01). Changes in retinal thickness (characteristic of retinal ischemia) were not observed in none of these time points as shown in Table 2. No clinical fundus abnormality was detected in the eyes from animals injected with HA or vehicle for 3 or 10 weeks (Figure 2).

Figure 3 depicts L-[3H]-glutamate uptake in retinas from animals handled and anesthetized once a week for 3 weeks but not injected in either eye, or from eyes treated with HA or vehicle for 3 or 10 weeks. The injection procedure itself did not affect this parameter. At 3 weeks of treatment, this parameter significantly decreased in eyes injected with HA as compared with vehicle-injected eyes (P<0.01). This difference was not evident in animals treated for 10 weeks. The effect of HA-induced ocular hypertension on glutamine synthetase activity is shown in Figure 4. At both time points, this enzymatic activity significantly decreased in retinas from eyes injected with HA (P<0.01). Table 3 shows the effect of ocular hypertension on retinal glutamine and glutamate release. For both glutamate and glutamine, about 50% of the preloaded radioactivity was released from retinal synaptosomal fractions during the efflux period in control conditions. At 3 weeks of treatment with HA, a significantly increase of basal but not of high K+ induced retinal L-[3H]-glutamine release was observed. The treatment with HA for 3 or 10 weeks did not modify L-[3H]-glutamate release. None of these parameters was affected by the injection procedure.

Glutamine uptake significantly increased in retinas from hypertensive eyes at 3 weeks but not at 10 weeks of treatment with HA (Figure 5). No differences in glutamine uptake were observed between non-injected and vehicle-injected eyes.

The effect of ocular hypertension on glutaminase activity was assessed in retinas from non-injected animals or from rats injected with HA or vehicle for the same periods. As shown in Figure 6, glutaminase activity significantly increased at 3 weeks but not at 10 weeks of treatment with HA (P<0.01). Glutaminase activity did not differ between eyes from non-injected animals and vehicle-injected eyes.

DISCUSSION

For the first time, the foregoing results (summarized in Figure 7) indicate a significant alteration of the retinal glutamate/glutamine cycle activity in rats exposed to experimentally elevated IOP. Glutamate is neurotoxic in excessive amounts. As no enzymes exist extracellularly that degrade glutamate, glutamate transporters are responsible for maintaining low synaptic glutamate concentrations. Present results demonstrate a significant decrease in retinal glutamate uptake in eyes injected with HA. In agreement, a significant reduction in the amount of the main retinal glutamate transporter (EAAT-1) assessed by Western blot analysis in a rat glaucoma model (28), and a down-regulation of this glutamate transporter in retinal Müller cells of glaucoma patients (29) were demonstrated. Although these studies did not assess changes in the functional capacity of glutamate transporters, our results demonstrated a removing glutamate disability in retinas from hypertensive eyes. Also in agreement with the work by Martin et al. (28), the decrease in


glutamate uptake in eyes injected with HA was transient, evident at only 3 weeks, but not 10 weeks, of treatment with HA.

The synaptically released glutamate is taken up into glial cells, where glutamine synthetase converts it into glutamine. Since Müller cells rapidly convert glutamate to glutamine, the driving force for glutamate uptake would be stronger in these cells than in neurons, which have much higher intracellular free glutamate concentrations (30). In fact, although glutamate uptake is controlled by the expression and post-translational modifications, physiological measurements suggest that glutamate uptake may also depend on its metabolism (31, 32). Indeed, an increase in internal glutamate concentrations significantly slows down the net transport of glutamate, and it was suggested that instantaneous intracellular glutamate metabolism could be needed for efficient glutamate clearance of the extracellular milieu (33, 34). The present results show a significant decrease in retinal glutamine synthetase activity in eyes injected with HA for 3 and 10 weeks. Therefore, this decrease in glutamine synthetase activity could account for the decrease in glutamate uptake. However, besides the coordination between both parameters, different mechanisms could also regulate their activity, since changes in glutamate uptake were only evident at 3 weeks of hypertension, while alterations in glutamine synthetase persisted at 10 weeks of treatment with HA.

Glutamine is released from Müller cells and could be a precursor for neuronal glutamate synthesis. The increase in the basal release and the uptake of glutamine in HA-treated eyes could provoke a raise in the availability of substrate for glutamate synthesis. Moreover, this increase in glutamate production could be potentiated further by the augment of glutaminase activity. The changes of glutamine influx and release as well as of glutaminase activity were evident only at 3 weeks of treatment but not thereafter. Although no changes in [3H]-glutamate release were observed, it is expected that a higher amount of endogenous glutamate could be released in the retina from hypertensive eyes, at least at 3 weeks of treatment with HA.

Decreasing the levels of expression of the transporter EAAT1 increases vitreal glutamate and is toxic to ganglion cells (35). Thus, the decrease in glutamate influx described herein could provoke an increase in synaptic glutamate levels. In addition, the changes in glutamine synthetase activity, glutamine uptake and release, as well as in glutaminase activity in retinas form hypertensive eyes could contribute synergically and/or redundantly to an excessive increase in synaptic glutamate levels.

Glutamate uptake and glutamine synthetase are mainly localized in Müller’s cells (36). The localization of glutamine transporters and glutaminase is ubiquitous (37), but free glutamine immunoreactivity is most abundant in ganglion cells and their axons (38). These evidences point at Müller and ganglion cells as putative targets for the ocular hypertension-induced by HA, albeit an effect on other cellular type(s) cannot be ruled out. As shown herein, no clinical fundus abnormality was observed in eyes injected with HA for 3 or 10 weeks. Thus, it seems that the increase in IOP generated by this model is unlikely to be high enough to occlude major retinal or choroidal vessels. Consistent with this conclusion, we found no significant changes in retinal thickness. The histological analysis showed a significant (≅40%) damage confined to the ganglion cell layer only in retinas from eyes injected with HA for 10 weeks, without evident changes in other retinal cells. Although glaucoma is known to cause primary death of ganglion cells, increasing evidences support that elevated IOP may also affect retinal glia (particularly,


Müller cells) even in the absence of pronounced morphologic changes in these cells. In fact, several reports support changes in Müller cells after short periods of ocular hypertension (39–41).

With the only exception of glutamine synthetase, the changes of glutamate/glutamine cycle parameters were transitory. Although this issue has no ready explanation, it is possible that these changes were provoked by a reversible injury to Müller or other retinal cells more than with cell death. The changes in glutamate recycling preceded functional and histological alterations induced by ocular hypertension (5, 6). Therefore, it is tempting to speculate that the changes in glutamate/glutamine cycle activity could be a causal factor in ocular hypertension-induced neuropathy. Furthermore, it seems possible that the increase in synaptic levels of glutamate could represent an initial (and probably reversible) insult responsible for initiation of damage that is followed by a slower secondary degeneration that ultimately results in cell death. We have shown a decrease in the retinal antioxidant defense system activity that mostly begins at 6 weeks of treatment with HA (8), and other authors have postulated that excessive levels of NO may contribute to this optic neuropathy (42). Based on these data, our working hypothesis is that retinal damage induced by ocular hypertension may result, at least in part, from oxidative stress induced by a glutamate/mediated pathway, as shown in other systems (43).

Increased IOP plays a causal, albeit not necessarily exclusive, role in glaucomatous visual loss. A therapy that prevents the death of ganglion cells is the main goal of treatment, although the current management of glaucoma is mainly directed at the IOP control. Taken together, these and previous results support that the impairment of glutamate neurotoxicity (44, 45), the decrease in NO levels (41), the manipulation of intracellular redox status using antioxidants (46), or preferably the combination of these treatments may be a therapeutic strategy to prevent glaucomatous cell death. Several lines of evidence support that melatonin is an effective retinal antioxidant (47, 48). In addition, we have shown that this methoxyindole is a potent inhibitor of the nitridergic pathway (49), and it increases glutamate uptake and glutamine synthetase activity and decreases glutaminase activity in the hamster retina (50). These results suggest that melatonin could be a promissory resource in the management of glaucoma since, by itself, it exhibits antioxidant and antinitridergic properties and may increase retinal glutamate clearance. Future experiments will address this hypothesis.

ACKNOWLEDGMENT

The authors wish to thank Diego Golombek and Pablo Arias for helpful discussion of this manuscript. This research was supported by the University of Buenos Aires and by the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina.

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Received October 28, 2004; accepted March 2, 2005.


Table 1 IOP values of rats injected with HA or vehicle for 3 or 10 weeks

IOP values (mm Hg) Time of treatment Vehicle HA 3 weeks 11.4 ± 0.3 21.3 ± 0.4** 10 weeks 11.3 ± 0.3 21.4 ± 0.8 **

IOP was assessed with a Tonopen XL in rats injected with HA in one eye and vehicle in the contralateral eye for 3 or 10 weeks. At these intervals, the injection of HA induced a significant increase in this parameter. No differences were observed in control eyes between 3 and 10 weeks of treatment. In animals handled and anesthetized once a week for 3 weeks, but not injected in either eye, IOP was 11.8 ± 0.4 (mean±SE, n=10 eyes). Data are mean ± SE (n=20 eyes/group) ** P < 0.01, by Student's t-test.


Table 2 Retinal thickness in animals injected with vehicle or HA for 3 or 10 weeks

Thickness (µm) 3 weeks

10 weeks

Vehicle

HA Vehicle HA

TOTAL 134 ± 6.2

130 ± 8 132 ± 7 129 ± 6.8

IPL 34.2 ± 5

33.2 ± 3.8 32.7 ± 4.6 31.6 ± 4.2

INL 24.7 ± 3.5

23.1 ± 2.5 25 ± 2.8 23.3 ± 2.3

ONL 38.3 ± 4

36.7 ± 5.2 37.1 ± 3 35.5 ± 4.7

The morphometric analysis of the thickness of the whole retina and retinal layers was assessed as described in Materials and Methods. No statistically significant difference was found in these parameters among animals injected with vehicle or HA for 3 or 10 weeks. Data are mean ± SEM

(n=5 animals per group). Total, the whole retina; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.


Table 3 Effect of ocular hypertension on [3H]-glutamine and [3H]-glutamate efflux.

[3H]-Glutamine release [3H]-Glutamate release

Vehicle AH Vehicle AH

Basal 59.6 ± 2.2 72.0 ± 3.0** 52.0 ± 2.5 52.3± 4.0 3 Weeks

High K+ 77.4 ± 3.0** 80.7 ± 2.2** 71.6 ± 5.2** 70.0 ± 4.1**

Basal 60.0 ± 3.3 62.0 ±3.4 54.2 ± 3.0 52.0 ± 4.0 10 Weeks

High K+ 77.4 ± 5.1** 78.0 ± 2.3** 70.3 ± 4.2** 68.5 ± 2.1**

High K+ (50 mM) induced a significant release of both [3H]-glutamine and [3H]-glutamate. The treatment with HA for 3 but not for 10 weeks significantly increased basal but not high K+- induced glutamine efflux. In eyes injected with HA for 3 or 10 weeks, no changes in basal or high K+-induced glutamate release were observed. In animals handled and anesthetized once a week for 3 weeks but not injected, the basal release of [3H]-glutamine was: 56 ± 3.0, whereas high K+-induced release was 81 ± 5.2. [3H]-glutamate release in these animals was 55 ± 4.2 and 68 ± 3.5 for basal and high K+, respectively. Data are mean ± SEM (n=10–12 animals/group). **P < 0.01 vs basal release, by Dunnet’s test.


Fig. 1

Figure 1. Representative histology of transverse retinal sections from rats in which one eye was injected with vehicle and the contralateral eye was injected with HA for 3 or 10 weeks. Note the diminution of ganglion cell layer (GCL) cells only in the eye injected with HA for 10 weeks. The other retinal layers showed a normal appearance. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Hematoxylin and eosin. Scale bar = 15µm.


Fig. 2

Figure 2. Representative fundus images from HA- or vehicle-injected eyes for 3 or 10 weeks. None of these eyes showed signs of impairment of blood flow, hemorrhages, or edema in the retina or choroid. In addition, the optic disc morphology was similar in shape and color in vehicle- and HA-injected eyes at both time points.


Fig. 3

Figure 3. Effect of intracameral injections of HA on retinal glutamate uptake. A significant decrease of glutamate influx was observed in retinas from eyes injected for 3 but not for 10 weeks with HA as compared with eyes injected with saline solution. No differences in this parameter were observed between animals handled and anesthetized once a week for 3 weeks but not injected (control) and animals injected with vehicle for the same period (vehicle). Data are mean ± SEM (n=10–12 animals per group). ** P < 0.01 by Student’s t-test.


Fig. 4

Figure 4. Retinal glutamine synthetase activity in eyes injected with HA or vehicle for 3 or 10 weeks and in non-injected eyes (control). At both time points, this enzymatic activity significantly decreased in eyes injected with HA as compared with vehicle-injected eyes. The injection procedure itself did not change glutamine synthetase activity. Shown are means ± SEM (n=10–12 animals/group). ** P < 0.01, by Student’s t-test.


Fig. 5

Figure 5. Effect of experimental ocular hypertension on retinal L-glutamine uptake. The treatment with HA for 3 weeks significantly increased retinal glutamine influx. At 10 weeks of treatment, no differences in glutamine influx were detected between HA- and vehicle-injected eyes. No differences between not injected and saline-injected animals were observed. Data are mean ± SEM (n=10–12 animals per group). ** P < 0.01 by Student’s t-test.


Fig. 6

Figure 6. Effect of intracameral injections of HA or vehicle on glutaminase activity in rat retina. At 3 weeks of treatment, a significant increase in the conversion of glutamine to glutamate was observed. The injection procedure itself did not change glutaminase activity. No differences were detected in animals injected with HA for 10 weeks. Data are mean ± SEM

values (n=10–12 animals per group). ** P < 0.01 by Student’s t-test.


Fig. 7

Figure 7. Schematic representation of the retinal glutamate/glutamine cycle and its modulation by the treatment with HA for 3 weeks. As shown, ocular hypertension induced by intracameral injections of HA decreased glutamate uptake and glutamine synthetase activity, whereas it increased glutamine uptake and release as well as glutaminase activity. Positive effects are noted by upward arrow; downward arrow indicates negative modulations.


Effect of glaucoma on the retinal glutamate/glutamine cycle activity

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