and eosin (H&E). The number of RGC was counted in six areas (25 mm 25 mm each) of each section (n ¼ 6 per eye) at 300 mm from the optic nerve head on the superior and inferior hemisphere, using light microscopy (40 magnification). The data were expressed as mean7SEM per area, and were evaluated statistically for differences using the Student’s t-test.

Microdialysis

Extracellular glutamate was monitored in the retina of anesthetized rats (urethane, 1500 mg kg–1, i.p.) during and after pressure-induced ischemia using a microdialysis technique. For implantation, a microdialysis probe (concentric design, 2 mm regenerated cellulose membrane, molecular weight cutoff 5 kDa) was implanted into vitreous chamber through the nonvascular pars plana region of the sclerotic coat after it had been punctured with a surgical needle (23 gauge). The surface of the dialysis membrane was secured perpendicularly to the retina for stable sampling during the experiment. Superfusion medium was continuously delivered via the probe at a rate of

2mL min–1. The composition of the medium (in mM)

was as follows: NaCl, 125; KCl, 2.5; MgCl2, 1.18; CaCl2, 1.26; NaH2PO4, 0.2; pH adjusted to 7.0. After

2h stabilization period, dialysate samples (20 mL) were collected at 10 min intervals before, during, and after ischemia. For analysis, the dialysate samples were derivatized with o-phthalaldehyde (OPA) and the concentration of glutamate determined as previously reported (Richards et al., 2000) by means of a high-performance liquid chromatography (HPLC) equipped with a fluorescence detector. Briefly, separation was achieved with a Hypersil ODS column (5 mm, 150 mm 3 mm, Chrompack, Milan, Italy) using a short methanol gradient (7–14% over 15 min) in 50 mM sodium acetate buffer, pH 6.95, followed by elution of remaining peaks with 95% methanol. Total run time was 17 min. The baseline concentration of glutamate was the mean concentration obtained by averaging the six samples collected consecutively at 10 min intervals immediately prior to the onset of ischemia and was used as control.

All experiments were carried out in accordance with the European Community Council Directive of November 24, 1986 (86/609/EEC). All efforts

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were made to minimize animal suffering and to use only the number of animals necessary to produce reliable results.

Drug application

For neuropathological studies, control animals (n ¼ 6) received injections of saline (1 mg kg 1, given i.p. twice daily), whereas test group received E2 (i.p., 0.2 mg kg 1; n ¼ 3) 30 min before ischemia or the estrogen receptor antagonist, ICI 182-780 (i.p., 0.2 and 2 mg kg 1; n ¼ 3 per group) 1 h before injection of E2.

For neurochemical studies, animals received systemic administration of E2 (i.p., 0.2 mg kg 1; n ¼ 5) and the 17a-isomer of estradiol (E2a, i.p., 0.2 mg kg 1; n ¼ 3) 30 min before ischemia. ICI 182-780 (i.p., 0.2 and 2 mg kg 1; n ¼ 5 and n ¼ 3, respectively) was administered 1 h before injection of E2.

E2, E2a, and ICI 182-780 were purchased from SIGMA (Italy).

Statistical analysis

All numerical data are expressed as the mean 7 SEM. Data were tested for statistical significance with paired Student’s t-test or by ANOVA followed by Dunnett’s test for multiple comparisons.

Results

17b-Estradiol pretreatment minimizes RGC loss

As shown in Table 1, 50 min of IOP-induced ischemia followed by 24 h of reperfusion caused a reduction in the number of RGCs by 28.03%. Systemic administration of E2 (0.2 mg kg 1), 30 min before ischemia, protected against RGC damage observed 24 h after delivery of the ischemic insult (Fig. 1) and significantly reduced the percentage loss of RGC to 7.14% (Table 1). A pretreatment with ICI 182-780, a specific estrogen receptor antagonist, failed to abrogate the neuroprotection afforded by E2 ( 6.63%) at the doses of 0.2 mg kg 1 (Table 1, Fig. 1), whereas it partially counteracted ( 15.18%) the effect of E2 at a dose of 2 mg kg 1 (Table 1).

High IOP-induced ischemia enhances extracellular glutamate in the retina: effect of 17b-estradiol

The time course of changes in extracellular glutamate during ischemia and reperfusion in rat (n ¼ 6) is illustrated in Fig. 2. The extracellular level of glutamate from the retina (1.08970.160 mM)

increased after the first 10 min of ischemia (2.03270.258 mM) with a larger and statistically significant increase observed at 10 and 150 min after the reperfusion had started (4.46570.746, po0.001 and 3.68371.158 mM, po0.05, respectively).

Systemic administration of E2 (0.2 mg kg 1 given i.p.; n ¼ 5 rats), 30 min before ischemia, did not

significantly affect the glutamate peak increase observed at 10 min after ischemia whereas it minimized the elevation of glutamate observed during the reperfusion period (Fig. 2). More importantly, E2 counteracted the glutamate increase observed after 10 and 150 min of reperfusion (2.40670.681 mM vs. basal levels 1.3187 0.307 mM, po0.05 and 1.22470.183 vs. basal levels 1.31870.307 mM, respectively) (Figs. 2 and 3). Pretreatment with the estrogen receptor antagonist ICI 182-780 (0.2 mg kg 1 given i.p.; n ¼ 5 rats) failed to counteract the effect on extracellular glutamate levels by E2 during reperfusion (Fig. 3), whereas at the dose of 2 mg kg 1 (n ¼ 3) it counteracted the effect of E2 at 10 min after reperfusion (data not shown). Interestingly, systemic administration, 30 min before ischemia, of E2a (0.2 mg kg 1, given i.p.; n ¼ 3 rats), which weakly binds to estrogen receptors, does not affect the glutamate peak observed at 10 min of reperfusion but, likewise to E2, counteracted the glutamate increase in the late reperfusion phase (Fig. 4).

Discussion

High IOP-induced ischemia is an established animal model to study the mechanisms underlying RGC death that also recapitulates features of acute angle closure glaucoma (Osborne et al., 2004). Recently, under these experimental conditions, we have reported that a delayed and progressive loss of viable cells in the RGC layer is observed starting from 6 h after the beginning of the reperfusion to peak at 7 days (Nucci et al., 2005). The mechanism underlying cell loss implicates overactivation of NMDA and non-NMDA subtypes of glutamate receptors and consequent accumulation of nitric oxide, being the loss minimized by systemic pretreatment with antagonists of the NMDA and non-NMDA receptors and by systemic pretreatment with l-NAME, an inhibitor of nitric oxide synthase (Nucci et al., 2005). The excitotoxic, glutamate-mediated, nature of the underlying mechanism of RGC death has also been confirmed by neurochemical data demonstrating that, during