Melatonin: a novel neuroprotectant for the treatment of glaucoma

Introduction

Glaucoma is a leading cause of blindness worldwidecharacterized by specific visual field defects because of theloss of retinal ganglion cells (RGC) and damage to the opticnerve head. Ocular hypertension is probably the most

important risk factor in primary angle open glaucoma, themore frequent form of glaucoma. It is estimated that half ofthose affected may be not aware of their condition because

symptoms may not occur during the early stages of thedisease. When vision loss appears, considerable andpermanent damage has already occurred. Medications

and surgery can help to slow the progression of someforms of the disease, but at present, there is no cure [1].

The value of animal models as substitutes for the study of

human pathology has been recognized by the scientificcommunity for decades. An experimental model system ofpressure-induced optic nerve damage would greatly facili-tate the understanding of the cellular events leading to

RGC death and how they are influenced by IOP and otherrisk factors associated with glaucomatous neuropathy. Wehave developed an experimental model of glaucoma in rats

through weekly injections of hyaluronic acid (HA) in theeye anterior chamber. Acute or chronic injections of HAsignificantly increase IOP when compared with the contra-

lateral vehicle-injected eye [2]. Moreover, chronic injectionsof HA for 6 or 10 (but not 3) weeks significantly decrease

the electroretinographic activity [3]. After 10 wk of ocularhypertension, a significant loss of RGC layer cells and opticnerve fibers was observed in eyes treated with HA [3]. These

results indicate that intracameral injections of HA mimiccentral features of human primary open-angle glaucoma.Besides ocular hypertension, several concomitant factors

may significantly contribute to the neurodegeneration (for

review [4, 5]). We have shown that chronic injections of HAprovoke a deficit in retinal glutamate clearance [6], asignificant upregulation of the retinal nitridergic pathway

activity [7], a decrease in the retinal gamma-aminobutyricacid (GABA)ergic activity [8], as well as a significantdecrease in the antioxidant defense system activity [9],

suggesting that alterations in glutamate, NO, and GABAlevels, as well as oxidative stress could be involved inglaucomatous neuropathy.There is a very large body of evidence documenting

melatonin as an antioxidant [10–13]. Not only melatonin,but also several of its metabolites generated during its freeradical scavenging action act as antioxidants. The kynure-

nic pathway of melatonin metabolism includes a seriesof radical scavengers with the possible sequence:Melatonin fi cyclic 3-hydroxymelatonin fi N1-acetyl-

N2-formyl-5-methoxykynuramine (AFMK) fi N1-acetyl-5-methoxykynuramine (AMK). In the metabolic step frommelatonin to AFMK, up to four free radicals can be

consumed [12]. Because of this pathway, melatonin�s

Abstract: Glaucoma is a leading cause of blindness. Although ocular

hypertension is the most important risk factor, several concomitant factors

such as elevation of glutamate and decrease in gamma-aminobutyric acid

(GABA) levels, disorganized NO metabolism, and oxidative damage could

significantly contribute to the neurodegeneration. The aim of this report was

to analyze the effect of melatonin on retinal glutamate clearance, GABA

concentrations, NO synthesis, and retinal redox status, as well as on

functional and histological alterations provoked by chronic ocular

hypertension induced by intracameral injections of hyaluronic acid (HA) in

the rat eye. In normal retinas, melatonin increased glutamate uptake,

glutamine synthase activity, GABA turnover rate, glutamic acid

decarboxylase activity, superoxide dismutase activity, and reduced

glutathione (GSH) levels, whereas it decreased NOS activity, L-arginine

uptake, and lipid peroxidation. To assess the effect of melatonin on

glaucomatous neuropathy, weekly injections of HA were performed in the eye

anterior chamber. A pellet of melatonin was implanted subcutaneously 24 hr

before the first injection or after six weekly injections of HA. Melatonin,

which did not affect intraocular pressure (IOP), prevented and reversed the

effect of ocular hypertension on retinal function (assessed by

electroretinography) and diminished the vulnerability of retinal ganglion cells

to the deleterious effects of ocular hypertension. These results indicate that

melatonin could be a promissory resource in the management of glaucoma.

Nicolas A. Belforte*, Marıa C.Moreno*, Nuria de Zavalıa, PabloH. Sande, Monica S. Chianelli,Marıa I. Keller Sarmiento andRuth E. Rosenstein

Laboratory of Retinal Neurochemistry and

Experimental Ophthalmology, Department of

Human Biochemistry, School of Medicine,

University of Buenos Aires, CEFyBO/

CONICET, Buenos Aires, Argentina

Key words: gamma-aminobutyric acid,

glaucoma, glutamate, melatonin, nitric oxide,

oxidative damage

Address reprint requests to Ruth E. Rosen-

stein, Departamento de Bioquımica Humana,

Facultad de Medicina, CEFyBO, UBA, Para-

guay 2155, 5�P, (1121), Buenos Aires,

Argentina.

E-mail: ruthr@fmed.uba.ar

*Both authors contributed equally to this work.

Received October 29, 2009;

accepted February 12, 2010.

J. Pineal Res. 2010; 48:353–364Doi:10.1111/j.1600-079X.2010.00762.x

� 2010 The AuthorsJournal compilation � 2010 John Wiley & Sons A/S

Journal of Pineal Research

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efficacy as an antioxidant is greatly increased. In addition,we have shown that melatonin is a potent inhibitor of thenitridergic pathway [14] and it increases glutamate uptake

and glutamine synthetase (GS) activity and decreasesglutaminase activity in the golden hamster retina [15]. Theaim of this report was to analyze the effect of melatonin onglutamate clearance, NO synthesis, GABA concentrations,

and the retinal redox status, as well as on functional andhistological alterations provoked by chronic ocular hyper-tension in the rat retina.

Materials and methods

Animals and Tissues

Male Wistar rats (average weight, 200 ± 40 g) were housedin a standard animal room with food and water ad libitumunder controlled conditions of humidity and temperature(21 ± 2�C), under a 12- hr light: 12 hr dark lighting

schedule (lights on at 07.00 hr). Animals were sacrificedby decapitation at 12.00 hr, eyeballs were quickly enucle-ated and corneas removed. Immediately after dissecting,

retinas were processed as described in the text for eachassay. One group of rats was subcutaneously implantedwith a pellet of melatonin (20 mg with 3% w/v vegetable oil

compressed in a cylinder of 2.5 mm diameter and 1 mmlength), while a control group was sham-operated withoutpellet implanting. The pellet of melatonin was implanted24 hr before the first injection or after six weekly injections

(3 days after the 6th injection) of HA, and it was replacedevery 15 days. All the experiments were conducted inaccordance with the Association for Research in Vision and

Ophthalmology Statement for the Use of Animals inOphthalmic and Vision Research.

L-3H-glutamate and L-3H-arginine uptake assessment

The influx of L-3H-glutamate or L-3H-arginine was

assessed in a crude synaptosomal fraction of rat retinas,as previously described [6, 7 respectively]. Two retinas(from the same animal) were pooled and homogenized (1:9w/v) in 0.32 m sucrose containing 1 mm MgCl2 and

centrifuged at 900 g for 10 min at 4� C. Nuclei-freehomogenates were further centrifuged at 30,000 g for20 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 lg protein/100 lL) were incubated with 100 lL of 3H-glutamate (10 lm, 500,000–800,000 dpm/tube, specificactivity 17.25 Ci/mmol) or 3H-arginine (10 lm, 800,000–1,000,000 dpm/tube, specific activity 53.4 Ci/mmol). After

5 min, aminoacids uptake was terminated by adding 4 mLof ice-cold HEPES-Tris buffer. The mixture was immedi-ately poured onto Whatman GF/B filters under vacuum.

The filters were washed twice with 4 mL aliquots of ice-coldbuffer and the radioactivity on the filters was counted in aliquid scintillation counter. Nonspecific uptake of 3H-

glutamate or 3H-arginine into synaptosomes was assessedby adding an excess of glutamate or arginine (10 mm),respectively.

Glutamine synthetase (GS) assessment

Each retina was homogenized in 200 lL of 10 mm potas-

sium phosphate, pH 7.2. GS activity was assessed asdescribed [6]. Reaction mixtures contained 150 lL ofretinal homogenates and 150 lL of a stock solution(100 mm imidazole-HCl buffer, 40 mm MgCl2, 50 mm

b-mercaptoethanol, 20 mm ATP, 100 mm glutamate, and200 mm hydroxylamine, adjusted to pH 7.2). Tubes wereincubated 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 TCA). Samples were placed for5 min on ice. Precipitated proteins were removed by

centrifugation, and the absorbance of the supernatantswas read at 535 nm against a reagent blank. Under theseconditions, 1 lmol of c-glutamylhydroxamic acid gives anabsorbance of 0.340. GS specific activity was expressed as

micromoles of c-glutamylhydroxamate per hour per milli-gram of protein.

Glutaminase activity assessment

Glutaminase activity was assessed as described [6]. Each

retina was homogenized in 100 lL of 0.1% Triton X-100 in7.5 mm Tris HCl, pH 8.8. The assay mixture contained30 lL of retinal homogenate (200–400 lg of proteins),

20 mm glutamine, 0.2- 0.5 lCi L-3H-glutamine, and 45 mm

potassium phosphate, pH 8.2, in a total volume of 100 lL.Tubes were incubated for 1 hr at 30�C, with gentleagitation. The reaction was stopped by adding 1 mL of

cold 20 mm imidazole, pH 7.0. Samples were brieflycentrifuged, and the supernatants were applied to 0.6 cm· 3.5 cm beds of anion exchange resin (Dowex, AG1-X2,

200-400 mesh hydroxide form, Bio-Rad Laboratories,Hercules, CA, USA) previously charged with 1 m HCland washed with water. The reaction substrate was

removed with 6 mL of imidazole buffer, which werediscarded and the reaction product was eluted with 3 mLof 0.1 m HCl. Aliquots of this fraction were mixed withscintillation cocktail for measurement of radioactivity.

Blank was determined from samples lacking retinal homo-genates. Glutaminase specific activity was expressed aslmoles of glutamate per milligram protein per hour.

Intravitreal injections of melatonin

Animals were anesthetized with ketamine hydrochloride(150 mg/kg) and xylazine hydrochloride (2 mg/kg)administered intraperitoneally. A drop of proparacaine

(0.5%) was administered in each eye for local anesthesia.With a Hamilton syringe (Hamilton, Reno, NV, USA)and a 30-gauge needle, 2 lL of sterile pyrogen-freesaline containing melatonin (final concentration 10 or

100 nm in vitreous cavity, considering a vitreous volumeof 60 lL [16]) were injected into one eye of anesthetizedrats, while an equal volume of vehicle was injected in

the fellow eye. Injections were applied at 1 mm of thelimbus and the needle was left in the eye for 60 s; thissmall volume prevented the increase in IOP and volume

loss.

Belforte et al.

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GABA turnover rate assessment

GABA turnover rate was measured as previously described

[17] in the retina from eyes intravitreally injected withmelatonin or vehicle. To prevent the postmortem increasein GABA content, animals were injected with the glutamicacid decarboxylase (GAD) inhibitor, 3-mercaptopropionic

acid (50 mg/kg i.p.) 2.5 min before sacrifice [18]. GABAturnover rate was assessed by the accumulation of GABAlevels following inhibition of GABA transaminase [19]. The

major assumption of the method used was that after theadministration of c-vinyl GABA (a gift from Merrel DowResearch Institute, Strasbourg, France), the accumulation

of GABA was linear for at least 1 hr. c-Vinyl GABA wasinjected i.p. at a 1 g/kg dose, 62.5 min before sacrifice.Retinas were excised, individually homogenized in distilledwater, and centrifuged at 12,000 g for 15 min, the pellets

being discarded. GABA concentrations were assessed by aradioreceptor assay [20] with a detection range for GABAof 2.5–100 pmol. To ascertain whether the displacement of3H-muscimol from its binding sites was because of endog-enous GABA, tissue samples were incubated with thehighly specific GABA-degrading enzyme system, GABAse,

before the assay. Either standard or endogenous GABAwas completely (i.e. more than 85%) degraded by GABAse.

Glutamic acid decarboxylase (GAD) activityassessment

GAD activity was determined as described [17] in the retina

from eyes intravitreally injected with vehicle or melatonin.Retinas were individually homogenized in 50 mm phosphatebuffer, pH 6.8. Aliquots of homogenate (0.2–0.4 mg protein)

were incubated in 1.5 mL-Eppendorf polypropylene tubesat 37�C for 1 hr. The incubation mixture (300 lL) contained1-14C-L-glutamic acid (0.2 lCi/mL, specific activity 49.6 Ci/

mmol), 1 mm L-glutamic acid, 200 lm pyridoxal phosphate,and 10 mm b-mercaptoethanol. Incubations were carriedout in triplicates. The reaction was stopped by adding 10%TCA, and the 14CO2 released during an additional 2-hr

period was captured on filter papers embedded in hyaminehydroxide. Blanks included all reagents except that TCAwas added before the homogenate.

NOS activity assessment

Retinal NOS activity was assessed as previously described[7]. Each retina was homogenized in 100 lL of buffersolution containing 0.32 m sucrose and 0.1 mm EDTA

(adjusted to pH 7.4 with Tris base). Reaction mixturescontained 50 lL of the enzyme source, 50 lL of a bufferstock solution (final concentrations: 10 mm HEPES, 3 mm

CaCl2, 1 mm NADPH, 5 lm FAD, 1 mm b-mercaptoeth-

anol, L-3H-arginine 5 lCi/mL, purity greater than 98%),

and 1 lm L-arginine. After incubation at 37�C for 30 min,the reaction was stopped by adding 200 lL of stop buffer

(50 mm HEPES, 10 mm EDTA, and 10 mm EGTA, pH5.5) and cooling the tubes for 5 min. The solution wasmixed with 600 lL of resin Dowex AG50W-X8 (Na+

form) to remove L-arginine and centrifuged at 10,000 g for5 min. L-3H-citrulline in the supernatant was quantified by

liquid scintillation counting. Nonenzymatic conversion ofL-3H-arginine to L-3H-citrulline was tested by addingbuffer instead of the enzyme source.

Superoxide dismutase (SOD) assay

Each retina was homogenized in 200 lL of 50 mm sodium

phosphate buffer, pH 7.4, and centrifuged at 900 g for 30 s.The SOD assay was performed as previously described [9].In brief, epinephrine undergoes auto-oxidation rapidly at

pH 10.0 to produce adrenochrome, a pink-colored productthat was assayed at 480 nm in kinetic mode using UV/VISspectrophotometer (Beckman DU 65; Beckman Instru-

ments, Inc., Fullerton, CA, USA). SOD inhibits the auto-oxidation of epinephrine. The rate of inhibition wasmonitored at 480 nm and the amount of enzyme requiredto produce 50% inhibition was defined as 1 unit of enzyme

activity. Total SOD activity was expressed as units permilligram protein.

Reduced glutathione (GSH) levels assessment

GSH levels in retinal tissue were assessed as described [9].

Each retina was homogenized in 120 lL of 50 mm potas-sium phosphate buffer, pH 7.4. Homogenates were incu-bated in the presence or absence of melatonin for 30 min at

37�C. Then, 100 lL of these samples were mixed with25 lL of 50% TCA plus 1 mm EDTA. After 5 min at 4�C,samples were centrifuged at 13,200 g for 2 min. Aliquots ofthe supernatants were mixed with 800 lL of 0.25 mg/mL

5,5¢-dithio-bis(2-nitrobenzoic acid) diluted in 0.5 m potas-sium phosphate buffer. Absorbance was recorded at412 nm. The range of the standard curves of reduced

GSH was 0.50–50 nmol.

Measurement of thiobarbituric acid reactivesubstances (TBARS) levels

TBARS levels in retinal tissue were analyzed as described[9]. Retinas were homogenized in 15 mm potassium buffer

plus 60 mm KCl, pH 7.2. The homogenate (300 lL) wasmixed with 75 lL 10% SDS and 1.4 mL 0.8% thiobarbi-turic acid dissolved in 10% acetic acid (pH 3.5). This

solution was heated to 100�C for 60 min. After cooling, theflocculent precipitate was removed by centrifugation at3200 g for 10 min. After addition of 1.0 mL water and

5.0 mL of n-butanol-pyridine mixture (15:1, vol/vol), themixture was vigorously shaken and centrifuged at 2000 gfor 15 min. The absorbance of the organic layer was

measured at an emission wavelength of 553 nm by using anexcitation wavelength of 515 nm with a Jasco FP 770fluorescence spectrophotometer (Japan Spectroscopic Co.Ltd., Tokyo, Japan). The range of the standard curves of

malondialdehyde bis-dimethyl acetal (MDA) was 10–2000 pmol. Results were expressed as nanomoles MDAper milligram protein.

Melatonin level assessment

Melatonin levels were assessed as previously described [9].Briefly, retinas were homogenized in 2 mL of 0.1 m HCl,

Therapeutic effect of melatonin in glaucoma

355

and melatonin was extracted from the whole homogenatewith 5 mL of dichloromethane. The organic phase waswashed twice with 2% NaHCO3 and distilled water.

Aliquots of 1.5 mL of the organic layer were dried undervacuum and stored at -20�C until the radioimmunoassaywas performed. The samples were resuspended in 100 lL ofbuffer (6 mm NaN3, 0.1 m KH2PO4, and 0.1% gelatin, pH

7.5) and then mixed with 3H-melatonin (20,000–24,000 dpm, specific activity 38.8 Ci/mmol) and 50 lL ofa melatonin antiserum kindly provided by Dr. Takashi

Matozaki (Laboratory of Biosignal Sciences, Institute forMolecular and Cellular Regulation, Gunma University,Japan). The mixture was incubated for 16 hr at 4�C. Thebound/free separation was performed by the dextranchar-coal method, and the radioactivity of supernatant wasmeasured by a liquid scintillation counter. Melatoninvalues were obtained from a melatonin standard curve,

with an assay limit sensitivity of 20 pg per tube.

Hyaluronic acid (HA) injections

Rats were anesthetized as previously described. With asyringe (Hamilton) and a 30-gauge needle, 25 lL of HA

(10 mg/mL in saline solution; catalog no. H1751; SigmaChemical Co., St. Louis, MO, USA) was injected into oneeye of anesthetized rats, and an equal volume of vehicle

(saline solution) was injected in the fellow (control) eye aspreviously described [2]. Briefly, the eyes were focusedunder a Colden surgical microscope with coaxial light. Theneedle moved through the corneoscleral limbus to the

anterior chamber with the bevel down. When the tip of thebevel reached the anterior chamber, the liquid progressivelyincreased the chamber�s depth, separating the needle from

the iris and avoiding needle-lens contact. Injections wereapplied at the corneoscleral limbus beginning at hour 12and changing the site of the next injection hourly, by

rotating the head to achieve better access to the limbus. Theinjections and IOP assessments were performed afterapplying one drop of 0.5% proparacaine hydrochloride toeach eye.

Electroretinography

The electroretinographic activity was assessed after six orten weekly injections of HA or vehicle, as previouslydescribed [3]. Briefly, after 6 hr of dark adaptation, rats

were anesthetized under dim red illumination. Phenyleph-rine hydrochloride and tropicamide were used to dilate thepupils, and the cornea was intermittently irrigated with

balanced salt solution to maintain the baseline recordingand to prevent keratopathy. Rats were placed facing thestimulus at a distance of 20 cm. All recordings werecompleted within 20 min and animals were kept warm

during and after the procedure. A reference electrode wasplaced through the ear, a grounding electrode was attachedto the tail, and a gold electrode was placed in contact with

the central cornea. A 15 W red light was used to enableaccurate electrode placement. This maneuver did notsignificantly affect dark adaptation and was switched off

during the electrophysiological recordings. Electroretino-grams (ERGs) were recorded from both eyes simulta-

neously and ten responses to flashes of unattenuated whitelight (5 ms, 0.2 Hz) from a photic stimulator (light-emittingdiodes) set at maximum brightness (20 cd s/m2 without

filter) were amplified, filtered (1.5-Hz low-pass filter, 1000high-pass filter, notch activated), and averaged (AkonicBIO-PC, Argentina). The a-wave was measured as thedifference in amplitude between the recording at onset and

the trough of the negative deflection, and the b-waveamplitude was measured from the trough of the a-wave tothe peak of the b-wave. Runs were repeated 3 times with

5 min-intervals to confirm consistency. Mean values fromeach eye were averaged, and the resultant mean value wasused to compute the group means a- and b-wave ampli-

tude ± SEM. The mean peak latencies and peak-to-peakamplitudes of the responses from each group of rats werecompared.

Histological evaluation

After 10 wk of treatment with vehicle or HA, rats were

sacrificed and their eyes were immediately enucleated,immersed for 24 hr in a fixative containing 4% formalde-hyde in 0.1 m phosphate buffer (pH 7.2) and embedded in

paraffin. Eyes were sectioned (5 lm) along the verticalmeridian through the optic nerve head. Microscopic imageswere digitally captured with a Nikon Eclipse E400 micro-

scope (illumination: 6-V halogen lamp, 20 W, equippedwith a stabilized light source) via a Nikon Coolpix s10camera. Sections were stained with H&E and analyzed bymasked observers. The number of cells in the RGC layer

was calculated along 100 lm for each section. No attemptwas made to distinguish cell types in the RGC layer forenumeration of cell number. Measurements (·400) were

obtained at 1 mm dorsal and ventral from the optic disc.For each eye, results obtained from four separate sectionswere averaged and the mean of five eyes was recorded as the

representative value for each group.

IOP assessment

A tonometer (TonoPen XL; Mentor, Norwell, MA) wasused to assess IOP in conscious, unsedated rats, asdescribed [2]. All IOP determinations were assessed by

operators who were blind to the treatment applied to eacheye. Animals were wrapped in a small towel and heldgently, with one operator holding the animal and another

making the readings. Five IOP readings were obtained fromeach eye by using firm contact with the cornea and omittingreadings obtained as the instrument was removed from the

eye. The mean of these readings was recorded as the IOPfor that eye on that day. Mean IOP from each rat wasaveraged, and the resultant mean IOP was used to computethe group mean IOP ± SEM. IOP measurements were

weekly performed at the same time each week (between11.00 and 12.00 hr) to correct for diurnal variations in IOP.

Results

Figure 1 depicts the in vitro effect of melatonin on

glutamate uptake, and GS and glutaminase activities.Melatonin significantly increased glutamate uptake and

Belforte et al.

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GS activity and decreased glutaminase activity. Thethreshold concentration for the effect of melatonin on GS

and glutaminase activities was 0.1 nm, while for glutamateuptake, it was 1 nm. To analyze the effect of melatonin onthe retinal GABAergic system, the effect of an intravitreal

injection of melatonin on GABA turnover rate and GADactivity was assessed. Melatonin significantly increasedboth parameters at an intravitreal concentration of 100 nm

(Fig. 2). Figure 3 shows the in vitro effect of melatonin onretinal NOS activity and L-arginine uptake. Melatoninsignificantly decreased NOS activity at every concentrationtested. In addition, melatonin decreased 3H-L-arginine

uptake with a threshold concentration of 1 nm.

To analyze the influence of melatonin on the retinalredox status, its effect on SOD activity, as well as on

reduced GSH and TBARS levels was examined (Fig. 4).Melatonin significantly increased SOD activity and GSHlevels, whereas it decreased TBARS levels (an index of lipid

peroxidation). The threshold concentration for SOD andTBARS levels was 1 nm, while for GSH, it was 10 nm. Noeffect of melatonin on catalase activity was observed (data

not shown). These results indicated that melatonin modu-lates the glutamatergic, GABAergic, nitridergic, and retinalredox status in an opposite manner to that previouslydescribed for ocular hypertension induced by HA. Thus,

the following experiments were performed to assess the

Fig. 1. Effect of melatonin on rat retinalglutamate uptake, glutamine synthetase(GS), and glutaminase activities. Retinaswere preincubated for 30 min in the pres-ence or absence of melatonin (0.1–10 nm).Then, tissues were homogenized and theseparameters were assessed as described inMaterials and methods. Melatonin signif-icantly increased glutamate uptake andGS activity, whereas it decreased gluta-minase activity. Data are mean ± SEM(n = 8–10). **P < 0.01, *P < 0.05 byDunnett�s test.

Fig. 2. Effect of melatonin on retinalGABA turnover rate and glutamic aciddecarboxylase (GAD) activity. Melatoninwas intravitreally injected in one eye,while the contralateral eye was injectedwith vehicle. Three hour after injections,animals were sacrificed and GABA turn-over and GAD activity were assessed asdescribed in Materials and methods. At aconcentration of 100 nm, melatonin sig-nificantly increased both parameters. Dataare mean ± SEM (n = 8–10 animals pergroup). **P < 0.01, by Dunnett�s test.

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357

ability of melatonin of preventing glaucomatous neuropa-thy. For this purpose, melatonin (20 mg) was administeredas a subcutaneous pellet. The levels of retinal melatoninwere assessed at several intervals after the pellet implanta-

tion, as shown in Fig. 5. The retinal content of melatoninsignificantly increased 1, 3, and 7 days after the adminis-tration of melatonin.

To analyze the effect of melatonin on the retinal damageinduced by ocular hypertension, a pellet of melatonin was

implanted 24 hr before the first injection of HA, injectionsof HA were repeated once a week and the pellet ofmelatonin was replaced every 15 days. Figure 6 shows theeffect of melatonin on the scotopic ERG from eyes injected

with vehicle or HA for 6 or 10 wk. At both time points,chronic injections of HA significantly decreased the ERG a-and b-wave amplitude when compared with vehicle-injected

eyes, while their latencies remained unchanged. The treat-ment with melatonin significantly prevented the electroret-

Fig. 3. Effect of melatonin on retinal NOS activity and L-arginine uptake. Retinas were preincubated for 15 min in the presence ofmelatonin (0.1–10 nm). Then, the tissues were homogenized and processed as described in Materials and methods. For L-arginine uptake,retinal synaptosomal fractions were preincubated with melatonin (0.1–10 nm) for 15 min prior to the transport assay. Melatonin signifi-cantly reduced retinal NOS activity at any concentration tested, while it significantly decreased L-arginine influx at 1 and 10 nm. Data arethe mean ± SEM (n = 8–10), *P < 0.05, **P < 0.01, by Dunnett�s test.

Fig. 4. Effect of melatonin on retinalsuperoxide dismutase (SOD) activity,glutathione (GSH) and thiobarbituric acidreactive substances (TBARS) levels. Reti-nas were preincubated for 30 min in theabsence or presence of melatonin (0.1–10 nm). Then, the tissues were homoge-nized and these parameters were assessedas described in Materials and methods.Melatonin significantly increasedSOD activity and GSH levels, while itdecreased TBARS levels. Data are themean ± SEM (n = 8–10), **P < 0.01,*P < 0.05, by Dunnett�s test.

Belforte et al.

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inographic dysfunction observed both at 6 and 10 wk ofocular hypertension. At 10 wk of ocular hypertensioninduced by HA, a significant decrease in the RGC layercell number was observed, whereas the presence of mela-

tonin significantly prevented the decrease in this parameter,as shown in Fig. 7. These results indicated that melatonin isable to significantly prevent functional and histological

alterations induced by ocular hypertension.To analyze the therapeutic effect of melatonin on

glaucomatous neuropathy, a pellet of melatonin was

implanted after 6 wk of ocular hypertension, and func-tional and histological studies were performed at 10 wk oftreatment with HA. As shown in Fig. 8, melatonin reversed

the decrease in the scotopic ERG a- and b-wave amplitudeinduced by HA injections. Moreover, the treatment withmelatonin, which did not affect the number of ganglion cellsin vehicle-injected eyes, reversed the decrease in RGC layer

cell number (Fig. 9). When administered before the firstinjection of HA, melatonin did not affect the increase inIOP induced by HA at 3, 6, or 10 wk of treatment with

vehicle or HA, as shown in Fig. 10. Similar results wereobtained when melatonin was implanted after 6 wk ofocular hypertension (data not shown).

Discussion

Glaucoma, a leading cause of irreversible blindness, is a

progressive neuropathy characterized by loss of vision as aresult of RGC death. At present, there are no effectiveneuroprotectants to treat this disorder. For the first time,

these results indicate that melatonin, which did not affectIOP, not only prevented but also reduced functional andhistological alterations provoked by chronic ocular hyper-

tension.Various ways to increase IOP in the rat eye, generally by

impeding the outflow of aqueous humor were developed

[21–23]. Several advantages support the glaucoma modelinduced by chronic injections of HA: (i) a highly consistenthypertension can be achieved, (ii) it may have a reasonablylong course, (iii) daily variations in IOP persist in HA-

injected eyes, (iv) in contrast to other models, in alllikelihood, HA does not impede the blood flow out of theeye, (v) topical glaucoma therapies applied acutely and at

the currently used doses significantly reduces the hyperten-sion induced by HA, and (vi) it is easy to perform [2].

Fig. 5. Retinal melatonin levels after the implantation of a sub-cutaneous pellet of melatonin (20 mg). The content of melatoninsignificantly increased 1, 3, and 7 days after the administrationof melatonin. Data are the mean ± SEM (n = 6), *P < 0.05,**P < 0.01, by Dunnett�s test.

(A) (B)

Fig. 6. Preventive effect of melatonin onthe retinal dysfunction induced by chronicocular hypertension. Eyes were injectedwith vehicle or hyaluronic acid (HA) for 6or 10 wk in the absence or presence ofmelatonin, and the electroretinogram(ERG) was assessed at both time points.At 6 (panel A) and 10 wk (panel B), HAinduced a significant decrease in ERG a-and b-wave amplitude when comparedwith vehicle-injected eyes, whereas thetreatment with melatonin significantlyprevented the effect of ocular hyperten-sion. Melatonin did not affect theseparameters in vehicle-injected eyes. Dataare the mean ± SEM (n = 8–10),**P < 0.01 versus vehicle-injected eyes a:P < 0.05, b: P < 0.01 versus HA-in-jected eyes, by Tukey�s test. Lower panel:(left) Representative scotopic ERG tracesfrom eyes injected with vehicle, HA, orHA in the presence of melatonin for 6 wk;similar treatments for 10 wk (right). Scalebars, x-axis = 30 ms, y-axis = 100 lV.

Therapeutic effect of melatonin in glaucoma

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Using this model, we have identified several ethiopath-ogenic mechanisms for glaucomatous neuropathy, such asan increase in glutamate synaptic concentrations [6] andNO levels [7], as well as a decrease in GABA concentra-

tions [8] and in the retinal antioxidant defense systemactivity [9]. These results indicate that in control retinas,melatonin modulated these parameters in an opposite way

to that induced by ocular hypertension, as shown in

Fig. 11. It should be noted that chronic injections of HAsignificantly decrease retinal melatonin levels [9]. Therefore,it is tempting to speculate about a causal relationshipbetween the fall in melatonin content and the alterations

observed on glutamate, GABA, NO, and retinal oxidativestatus.We have previously demonstrated the effect of melatonin

on glutamate clearance [15] and NO production [14] in the

(A) (B) (C)

Fig. 7. Light micrographs of transverse sections of rat retinas from eyes injected with vehicle (A) or hyaluronic acid (HA) during 10 wk inthe absence (B) or presence of melatonin (C). In the eye injected with HA, a diminution of retinal ganglion cells (RGC) layer cells wasobserved, whereas melatonin, which did not affect this parameter in vehicle-injected eyes (data not shown), significantly prevented cell loss.The other retinal layers showed a normal appearance in all the experimental groups. IPL, inner plexiform layer; INL, inner nuclear layer;OPL, outer plexiform layer; ONL, outer nuclear layer. H&E. Scale bar: 100 lm. Lower panel: Data are mean number of RGC cell layer cells/100 lm ± SEM (n = 5). **P < 0.01 versus vehicle, a: P < 0.05 versus HA-injected eyes, by Tukey�s test.

Fig. 8. Therapeutic effect of melatonin on the retinal dysfunction induced by chronic ocular hypertension. After six weekly injections ofhyaluronic acid (HA), a pellet of melatonin was implanted subcutaneously. The injections of HA were repeated once a week and the scotopicelectroretinogram (ERG) was assessed at 10 wk of treatment with HA. Left panel: average amplitudes of scotopic ERG a- and b-waves.Ocular hypertension significantly decreased the ERG a- and b-wave amplitude, whereas the treatment with melatonin significantly reversedthe effect of ocular hypertension. Data are the mean ± SEM (n = 8–10), **P < 0.01 versus vehicle-injected eyes; a: P < 0.05, versus HA-injected eyes, by Tukey�s test. Right panel: representative scotopic ERG traces from eyes injected with vehicle, HA, or HA in the presence ofmelatonin. Scale bars: x-axis = 30 ms, y-axis = 100 lV.

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360

golden hamster retina. These results further confirm itseffect on these parameters in the rat retina. In agreement,melatonin was shown to decrease NO production in otherneural structures [24–26]. However, while the inhibitory

effect of melatonin on NOS activity in those tissues wasevident up to 1 nm, a higher sensitivity to the methoxyin-dole was evident in the rat retina, as it was effective even at

0.1 nm, suggesting that retinal NOS could be more suscep-

tible to melatonin than the enzyme from other tissues.Moreover, melatonin decreased the intracellular availabilityof NOS substrate (another limiting step in NO biosynthe-sis), as shown by its effect on L-arginine uptake.

Melatonin may be an effective cytoprotective agentagainst glutamate excitotoxicity in central nervous systeminjuries and diseases [27]. An appropriate clearance of

synaptic glutamate is required for the normal function ofretinal excitatory synapses and for prevention of neurotox-icity. Through its effect on glutamate uptake, GS, and

glutaminase activity, melatonin could decrease glutamatesynaptic concentrations, and therefore induce neuroprotec-tion against excitotoxic injury.

These results indicate that melatonin increased GABAlevels, as shown by its effect on GABA turnover rate andGAD activity. The effect of melatonin on the GABAergicactivity seems not to be exclusive for the retina as it was

previously demonstrated that melatonin increases GABAturnover rate and GAD activity in rat hypothalamus,cerebellum, and cerebral cortex [28, 29].

(A) (B) (C)

Fig. 9. Effect of melatonin on retinal ganglion cells (RGC) loss induced by chronic ocular hypertension. Melatonin was administered at6 wk of ocular hypertension induced by hyaluronic acid (HA). At 10 wk of ocular hypertension, a significant decrease in the number ofRGC was observed in HA-injected eyes (B), when compared with vehicle-injected eyes (A). The treatment with melatonin (C) significantlyreversed the effect of ocular hypertension induced by HA. H&E. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiformlayer; ONL, outer nuclear layer. H&E. Scale bar: 100 lm. Lower panel: Data are mean number of RGC cell layer cells/100 lm ± SEM(n = 5). **P < 0.01 versus vehicle, b: P < 0.01 versus HA-injected eyes, by Tukey�s test.

Fig. 10. Effect of melatonin on rat intraocular pressure (IOP). Eyeswere weekly injected with vehicle or hyaluronic acid (HA) for 3, 6,or 10 weeks, in the absence or presence of melatonin and IOP wasassessed 3 days after the last injection in each case. At all thesetime-points, melatonin did not affect IOP in vehicle- or HA-in-jected eyes. Data are the mean ± SEM (n = 8–10). **P < 0.01versus vehicle-injected eyes.

Fig. 11. Schematic representation of the retinal glutamate, NO,GABA levels and oxidative damage and their modulation byglaucoma and melatonin. Glaucoma increased glutamate and NOlevels, and oxidative damage, whereas it decreased GABA levels.Melatonin modulated all this parameters in an opposite manner.Positive effects are noted as , whereas indicates negativemodulations.

Therapeutic effect of melatonin in glaucoma

361

Excitotoxicity is a common pathogenic mechanism inneurodegenerative diseases, including probably glaucoma[5, 30], which may result from the failure of normal

compensatory antiexcitatory mechanisms necessary tomaintain cellular homeostasis. An abnormal glutamateoutflow may play a crucial role in triggering cellularevents leading to excitotoxic neuronal death. We have

previously demonstrated that ocular hypertension inducesa retinal GABAergic dysfunction [8], which could con-tribute to this imbalance. Because of this, ocular hyper-

tension may greatly shift the retinal excitation/inhibitionbalance and melatonin could restore the neural equilib-rium.

Melatonin has ubiquitous actions as a direct as well asan indirect antioxidant and free radical scavenger. Mel-atonin�s functions as an antioxidant include: (i) direct freeradical scavenging [10, 13], (ii) stimulation of antioxida-

tive enzymes [31], (iii) increasing the efficiency of mito-chondrial oxidative phosphorylation and reducingelectron leakage (thereby lowering free radical generation)

[32], and (iv) augmenting the efficiency of other antiox-idants [13].The retina is particularly susceptible to oxidative stress

because of its high consumption of oxygen, its highproportion of polyunsaturated fatty acids, and its directexposure to light. Melatonin decreases lipid peroxidation of

polyunsaturated fatty acids located in reactive oxygenspecies membranes [33], it protects retinal pigment epithe-lial cells from oxidative stress [34], it reduces NO-inducedlipid peroxidation in rat retinal homogenates [35], and it

prevents retinal oxidative damage from ischemia-reperfu-sion injury [36]. Although no changes in rat retinal catalaseactivity were observed in the presence of melatonin, it

significantly increased total SOD activity and GSH levelsand it reduced retinal lipid peroxidation. Hence, melatonincould provoke an impairment of glutamate neurotoxicity, a

decrease in NO levels, an elevation in GABA concentra-tions, and reduce oxidative stress [37]. Thus, the use ofmelatonin could be a therapeutic strategy to prevent

glaucomatous cell death. As shown herein, the chronictreatment with melatonin prevented functional alterationsand diminished the vulnerability of RGC to the deleteriouseffects of ocular hypertension. Most of the melatonin effects

shown in this report occurred with concentrations ofmelatonin in the low nanomolar range (0.1–1 nm), whichis compatible with high-affinity melatonin receptors

(�100 pm) [38]. This high sensitivity of melatonin suggeststhat the indole modulates these retinal parameters probablythrough receptor-mediated mechanism(s). Notwithstand-

ing, several antioxidant effects of melatonin were shown tobe nonreceptor-mediated events [13]. Therefore, it seemslikely that the effects of melatonin described herein couldinvolve both receptor and nonreceptor-mediated mecha-

nisms.Although melatonin conferred neuroprotection in the

experimental model of glaucoma induced by HA, the

translational relevance of this result is limited by the factthat melatonin was administered before the induction ofocular hypertension (e.g., 24 hr before the first injection of

HA). Therefore, additional experiments were performed totest whether melatonin could not only prevent but also

reduce glaucomatous neuropathy. These results indicatedthat the delayed treatment of eyes with ocular hyperten-sion resulted in similar protection when compared to eyes

treated from the onset of ocular hypertension. We do nothave any clear explanation for these results. In previousreports, we showed that an increase in synaptic glutamateconcentrations [6] and an increase in NO production [7] as

well a GABAergic dysfunction [8] occurred mostly priorto 6 wk of treatment with HA. Thus, it seems possiblethat alterations in glutamate, NO, and GABA may trigger

an initial insult responsible for initiation of damage that isfollowed by a slower secondary degeneration that ulti-mately results in cell death. In that sense, we showed that

oxidative stress is a longer lasting phenomenon that canbe observed even at 10 wk of ocular hypertension [9]. Inthis scenario, the preventive effect of melatonin (shown bythe administration of melatonin before the first injection

of HA) could be explained by the decrease in glutamateand NO levels, and an increase in GABA concentrations,while its therapeutic effect (shown by the administration

of melatonin at 6 wk of ocular hypertension) can beexplained essentially by its antioxidant effect. In thiscontext, the fact that melatonin was similarly effective in

the chronically treated animals and the delayed treatmentcould support the hypothesis that in both cases melatoninis able to reverse oxidative damage, which could be a key

factor in glaucomatous dysfunction and cell death.The effect of melatonin on IOP is still controversial.

Several studies did not find any effect of topical application,intravenous or intravitreal injection, and intraarterial

infusion of melatonin on rabbit IOP [39, 40], whereas othergroups have demonstrated that oral or topical administra-tion of melatonin reduces IOP in rabbits [41], monkeys [42],

and humans [43]. In our experimental setting, a subcuta-neous pellet of melatonin did not change IOP along 10 wkof treatment with HA. Currently, we do not have any

explanation for this discrepancy. However, differences inspecies (rats in our case), administration route, or dosecould account for it.

It has been suggested that neuroprotection in glaucomaimplies the use of drugs or chemicals to slow downwhatever causes loss of vision (the death of ganglion cells)without influencing IOP. To be effective, a neuroprotectant

must reach the optic nerve head and/or ganglion cells andwill therefore probably have to be taken orally. Subcuta-neously administered, melatonin reached the retina,

increasing the local levels of the methoxyindole. Becauseit will reach other parts of the body, any side-effect of anappropriate neuroprotectant must be reduced to a mini-

mum. In addition, ganglion cells are induced to die bydifferent triggers in glaucoma, suggesting that neuropro-tectants with multiple modes of actions are likely beeffective in the therapeutic management of glaucoma [44].

As shown herein, melatonin seems to fulfill all the require-ments to be included in the armamentarium of ophthalmictherapeutic resources, particularly as a neuroprotectant for

glaucoma treatment. Alone or combined with an ocularhypotensive therapy, a treatment with melatonin, a verysafe compound for human use, could be a new therapeutic

tool helping the challenge faced by ophthalmologiststreating glaucoma.

Belforte et al.

362

Acknowledgement

This research was supported by grants from the Agencia

Nacional de Promocion Cientıfica y Tecnologica (AN-PCyT), the University of Buenos Aires, CONICET,Argentina, and the John Simon Guggenheim Memorial

Foundation.

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