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17β-Estradiol prevents retinal ganglion cell loss induced by acute rise of intraocular pressure in rat
C. Nucci et al. (Eds.) Progress in Brain Research, Vol. 173 ISSN 0079-6123 Copyright r 2008 Elsevier B.V. All rights reserved
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17b-Estradiol prevents retinal ganglion cell loss induced by acute rise of intraocular pressure in rat Rossella Russo1, Federica Cavaliere1, Chizuko Watanabe2, Carlo Nucci3,4, Giacinto Bagetta1,5, Maria Tiziana Corasaniti4,6, Shinobu Sakurada2 and Luigi Antonio Morrone1,5, 1
Department of Pharmacobiology, University of Calabria, 87036 Arcavacata di Rende, Italy Department of Physiology and Anatomy, Tohoku Pharmaceutical University, Sendai, Japan 3 Physiopathological Optics, Department of Biopathology, University of Rome ‘‘Tor Vergata,’’ 00133 Rome, Italy 4 ‘‘Mondino-Tor Vergata’’ Center for Experimental Neurobiology, University of Rome ‘‘Tor Vergata,’’ 00133 Rome, Italy 5 Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University Center for Adaptive Disorders and Headache (UCADH), University of Calabria, 87036 Arcavacata di Rende, Italy 6 Department of Pharmacobiological Sciences, University ‘‘Magna Graecia’’ of Catanzaro, 88100 Catanzaro, Italy 2
Abstract: Glaucoma, is a progressive optic neuropathy often associated with increased intraocular pressure (IOP) and characterized by progressive death of retinal ganglion cells (RGCs). High acute rise of IOP is a model for retinal ischemia and may represent a model of acute angle closure glaucoma. Here we have used this experimental model in combination with a neurochemical and neuropathological approach to gain more insight in the neuroprotective profile of 17b-estradiol (E2), a steroid hormone, which has been shown to increase the viability, survival, and differentiation of primary neuronal cultures from different brain areas including amygdala, hypothalamus, and neocortex. Our data demonstrate that systemic administration of E2 significantly reduces RGC loss induced by high IOP in rat. In addition, pretreatment with E2, 30 min before ischemia, minimizes the elevation of glutamate observed during the reperfusion period. These effects seem to be in part mediated by the activation of the estrogen receptor, since a pretreatment with ICI 182-780, a specific estrogen receptor antagonist, partially counteracts the neuroprotection afforded by the estrogen. Keywords: glaucoma; excitotoxicity; oxidative stress; estrogens; microdialysis; neuroprotection
intraocular pressure (IOP) that is characterized by progressive death of retinal ganglion cells (RGCs) and consequent deterioration of the visual field. Several studies suggest that excitotoxicity occurs during retinal ischemia with subsequent loss of RGCs and that this process plays a role in the pathogenesis of ischemic retinopathy (Nucci et al., 2005; Casson, 2006). Glutamate functions as the major excitatory amino acid neurotransmitter
Introduction Glaucoma, one of the leading causes of blindness in the world (Quigley, 1996), is a progressive optic neuropathy often associated with increased
Corresponding author. Tel.: +39 0984 493054; Fax: +39 0984 493462; E-mail: [email protected]
DOI: 10.1016/S0079-6123(08)01144-8
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in the retina, but at high concentrations it becomes neurotoxic. The pivotal role of excessive glutamate in the mechanisms of retinal damage is also documented by the evidence that NMDA and non-NMDA receptor antagonists afford protection in experimental models of RGC death, both in vitro and in vivo (Adachi et al., 1998; Joo et al., 1999; Osborne et al., 1999; Nucci et al., 2005). Growing evidence also supports that oxidative stress is the leading mechanism of excitotoxic, glutamateinduced RGC loss in vitro (Luo et al., 2001) and in vivo (Nucci et al., 2005). Not only the interference with glutamate transmission may stem from the decrease of the retinal glutamate transport (Muller et al., 1998), but also glutamine synthetase, which converts glutamate to glutamine, is oxidatively modified in ocular hypertensive eyes (Tezel et al., 2005). In addition, retinal glutamate damage has been shown to be mediated in part through nitric oxide, a highly reactive radical species (Nucci et al., 2005). Based on previous observations, neuroprotection of RGC from excitotoxicity is becoming an important approach of glaucoma therapy. Interestingly, it has been recently demonstrated that 17b-estradiol (E2) minimizes RGC loss in DBA/2J mouse, an in vivo model of an inherited (pigmentary) glaucoma (Zhou et al., 2007), shows neuroprotective effect on axotomy-induced RGC death (Nakazawa et al., 2006), and protects RGC against glutamate cytotoxicity (Kumar et al., 2005). In addition, several studies showed that estrogens, a family of cholesterol-derived steroid hormones, are protective against various oxidative stress insults including excitotoxicity (Goodman et al., 1996; Singer et al., 1996, 1999; Weaver et al.,1997; Zaulyanov et al., 1999). Cumulative evidence from basic science and clinical research suggests that estrogens play a significant neuromodulatory and neuroprotective role in the brain, and this underlies their ability to ameliorate symptoms and decrease the risk of neurodegenerative conditions such as cerebrovascular stroke, Alzheimer’s disease, and Parkinson’s disease (see Amantea et al., 2005). The mechanisms underlying estrogen neuroprotection have not been completely elucidated and several mechanisms have been proposed to explain the neurotrophic and neuroprotective actions of estrogens, including modulation of synaptogenesis,
protection against apoptosis, anti-inflammatory activity, and increased cerebral blood flow (Garcia-Segura et al., 2001; Wise, 2003; Maggi et al., 2004). Using high IOP experimental model in rat, in combination with a neurochemical and neuropathological approach, we now report the neuroprotection afforded by systemic treatment with E2, showing that this hormone is able to prevent the glutamate-induced loss of RGC under these experimental conditions.
Methods Male, Wistar rats (250–300 g) (Charles River, Lecco, Italy) were maintained on a 12-h light–dark cycle. Before ischemia was induced, animals were anesthetized with chloral hydrate (400 mg kg–1, intraperitoneally (i.p.)). Corneal analgesia was achieved using topical drops of oxibuprocaine 0.4% (Novesina, Novartis Farma, Italy). Pupillary dilation was maintained using 0.5% tropicamide (Visumidriatic 0.5%, Visufarma, Italy). The anterior chamber of the right eye was cannulated with a 27-gauge infusion needle connected to a 500 mL plastic container of sterile saline, the IOP was raised to 120 mmHg for 50 min by elevating the saline reservoir. Retinal ischemia was confirmed by observing whitening of the iris and loss of the red reflex of the retina. Sham procedure was performed without the elevation of the bottle in control rats. Morphometric analysis Rats receiving ischemic insult or sham procedure were anaesthetized (chloral hydrate 400 mg kg–1, i.p.) and perfused through the left ventricle of the heart with 50 mL of heparinized phosphate buffered (pH 7.4) saline followed by 50 mL of 4% paraformaldehyde in phosphate buffered saline at 6 h, 12 h, 24 h, 48 h, and 7 days after reperfusion (n ¼ 6 per group). Two hours after the perfusion procedure had been completed, the eyes were enucleated and post-fixed in 4% paraformaldehyde for 72 h. Serial coronal sections, cut along the vertical meridian of the eye passing through the optic nerve head, were stained with hematoxylin
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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 2 mL 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 2 h 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
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 kg1, given i.p. twice daily), whereas test group received E2 (i.p., 0.2 mg kg1; n ¼ 3) 30 min before ischemia or the estrogen receptor antagonist, ICI 182-780 (i.p., 0.2 and 2 mg kg1; 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 kg1; n ¼ 5) and the 17a-isomer of estradiol (E2a, i.p., 0.2 mg kg1; n ¼ 3) 30 min before ischemia. ICI 182-780 (i.p., 0.2 and 2 mg kg1; 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 kg1), 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 kg1 (Table 1, Fig. 1), whereas it partially counteracted (15.18%) the effect of E2 at a dose of 2 mg kg1 (Table 1).
586 Table 1. Retinal ganglion cell (RGC) loss induced by acute high intraocular pressure is prevented by systemic treatment with 17bestradiol Experimental conditions
Number of RGC
Percentage vs. control
Control (sham-operated) Ischemia 17b-Estradiol (0.2 mg kg1)+ischemia ICI 182-780 (0.2 mg kg1)+17b-estradiol+ischemia ICI 182-780 (2 mg kg1)+17b-estradiol+ischemia
35.4370.08 25.5070.29# 32.9070.82 33.0870.32,y 30.0570.22
28.03 7.14 6.63 15.18
Elevated IOP-induced ischemia for 50 min was followed by a 24-h reperfusion period. Control animals (n ¼ 6) received injections of saline (1 mg kg1, given i.p. twice daily), whereas test group received i.p. 17b-estradiol (E2, n ¼ 3) 30 min before ischemia or the estrogen receptor antagonist, ICI 182-780 (i.p., n ¼ 3 per group), 1 h before injection of E2. Cell counting was performed in the ganglion cell layer of ischemic/reperfused and sham-operated rat retinas stained with hematoxylin and eosin. The number of RGC was counted in six areas of each section (n ¼ 6 for eye) using light microscopy. The data were expressed as mean7SEM per area, and were evaluated statistically for differences using the Student’s t-test. # p ¼ 0.000 vs. control. po0.01 vs. ischemia. y p ¼ 0.8 vs. E2+ischemia
Fig. 1. Retinal ischemia for 50 min followed by 24 h reperfusion reduces the number of cells in the retinal ganglion cell layer (B, n ¼ 6 rats) as compared to sham-operated rats (A, n ¼ 6). Systemic treatment with 17b-estradiol (C, i.p., 0.2 mg kg1, n ¼ 3 rats) prevents the tissue damage observed in (B). A pretreatment with ICI 182-780 (D, i.p., 0.2 mg kg1, n ¼ 3 rats), a specific estrogen receptor antagonist, failed to abrogate the neuroprotection afforded by 17b-estradiol. H&E staining. RGC: retinal ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer.
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 kg1 given i.p.; n ¼ 5 rats), 30 min before ischemia, did not
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Fig. 2. Neurochemical data obtained by intraocular microdialysis experiments in anesthetized rats (n ¼ 6) demonstrate that ischemia/ reperfusion insult increases intraretinal glutamate. The extracellular level of glutamate (GLU, solid line) from the retina tended to increase after the first 10 min of ischemia with a larger and statistically significant increase observed at 10 and 150 min after the reperfusion had started. Systemic administration of 17b-estradiol (i.p., 0.2 mg kg1, n ¼ 5 rats, dashed line), 30 min before ischemia, did not significantly affect the GLU peak increase observed at 10 min after ischemia, whereas it minimized the elevation of GLU observed during the reperfusion period. The baseline concentrations of GLU were the mean concentrations obtained by averaging the six samples collected consecutively at 10 min intervals immediately prior to the onset of ischemia and were used as basal values. Glutamate values (mM) are expressed as mean7SEM. Statistical significance was assessed by ANOVA followed by Dunnett’s test for multiple comparisons # and po0.05 vs. basal values; po0.001 vs. basal values.
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 kg1 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 kg1 (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 kg1, 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
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Fig. 3. Effect of 17b-estradiol (i.p., 0.2 mg kg1, n ¼ 5 rats) and of the estrogen receptor antagonist ICI 182-780 (0.2 mg kg1, n ¼ 5 rats) on levels of glutamate (GLU) observed at 10 and 150 min after reperfusion had started. Administration of 17b-estradiol (gray columns) significantly reduced the GLU increase observed after 10 and 150 min of reperfusion. ICI 182-780 (black columns) failed to counteract the effect on extracellular GLU levels afforded by 17b-estradiol. The white columns show the extracellular GLU levels obtained in ischemia/reperfusion (isch/rep) experiments (n ¼ 6). Data are expressed as mean7SEM percentage of basal values of GLU. The baseline concentrations of glutamate were the mean concentrations obtained by averaging the six samples collected consecutively at 10 min intervals immediately prior to the onset of ischemia. Data were tested for statistical significance with paired, two-tailed, Student’s t-test. po0.05 vs. isch/rep.
Fig. 4. Neurochemical data obtained by intraocular microdialysis experiments in anaesthetized rats (n ¼ 6) demonstrate that the extracellular level of glutamate (GLU, solid line) from the retina tended to increase after the first 10 min of ischemia with a larger and statistically significant increase observed at 10 and 150 min after the reperfusion had started. Systemic administration of 17a-estradiol (i.p., 0.2 mg kg1, n ¼ 3 rats, dashed line), 30 min before ischemia, did not significantly affect the GLU peak increase observed at 10 min after ischemia and after 10 min of reperfusion, whereas it minimized the elevation of GLU observed during the late reperfusion period. The baseline concentrations of GLU were the mean concentrations obtained by averaging the six samples collected consecutively at 10 min intervals immediately prior to the onset of ischemia and were used as basal values. Glutamate values (mM) are expressed as mean7SEM. Statistical significance was assessed by ANOVA followed by Dunnett’s test for multiple comparisons # and po0.05 vs. basal values; po0.001 vs. basal values.
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reperfusion, extracellular glutamate increases significantly in the retina of the ischemic eye and this is sensitive to the prevention afforded by systemic MK801 (Nucci et al., 2005). The present study shows that systemic pretreatment with E2, 30 min before ischemia, prevents glutamate increase during reperfusion and this is accompanied by minimization of RGC death at 24 h of reperfusion. Accordingly, recent data demonstrated that E2 minimizes RGC loss in DBA/2J mouse, an in vivo model of an inherited glaucoma (Zhou et al., 2007), has neuroprotective effect on axotomy-induced RGC death (Nakazawa et al., 2006), and protects RGC against glutamate cytotoxicity (Kumar et al., 2005). Here we monitored in vivo extracellular glutamate levels from rat retina during and after pressure-induced ischemia using an established microdialysis technique (Nucci et al., 2005). An increase of dialysate glutamate levels occurred during ischemia, followed by a more pronounced and statistically significant increase during the reperfusion phase. Interestingly, neurochemical data show that pretreatment with E2 fails to counteract glutamate increase typically observed during ischemia, while it significantly inhibits the subsequent increase observed during reperfusion. The mechanism underlying neuroprotection afforded by E2 is not known; however, under our experimental conditions, the effect of E2 on RGC seems to be mediated in part by the activation of the estrogen receptors. In fact, a pretreatment with high concentrations of a specific estrogen receptor antagonist, the compound ICI 182-780, partially counteracts the neuroprotection afforded by the estrogen. Interestingly, this high dose of ICI 182-780 is able to counteract partially the effect of E2 on extracellular levels of glutamate during reperfusion period, whereas a lower dose of ICI 182-780 fails to counteract any effect of E2 on extracellular glutamate levels during reperfusion. Particularly, the higher dose of ICI 182-780 prevents the effect of E2 in the early reperfusion phase, but not during the late phase. This experimental observation seems to suggest that in our experimental conditions, the increase of glutamate observed in the early phase of reperfusion is under the control of estrogen receptors whereas the accumulation of
glutamate in the late reperfusion phase seems to stem from an estrogen receptor-independent mechanism. Accordingly, we demonstrate that the E2a, a weak estrogen receptor agonist (Clark and Markaverich, 1983; Lubahn et al., 1985, Yang et al., 2003), does not affect the glutamate increase observed in the early phase of reperfusion but it is effective as E2 in minimizing the glutamate increase observed in the late reperfusion period. The latter result strengthens the hypothesis that this estrogenic effect is mediated via a mechanism that does not require binding to the cytosolic estrogen receptor. Interestingly, although many of the estrogen neuroprotective effects appear to be mediated through the activation of intracellular estrogen receptors (ER), ERa and ERb, several observations support the possibility that estrogens exert their potent neuroprotective effects through a mitochondrial mechanism (see Simpkins and Dykens, 2008). Increasing experimental evidence implicates failure of mitochondrial energy metabolism in the pathogenesis of neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases (see Tezel, 2006). Following a detrimental stimulus, estrogens preserve ATP production, prevent production of ROS, moderate excessive cellular and mitochondrial Ca2+ loading, and preserve mitochondrial membrane potential (see Simpkins and Dykens, 2008). Accordingly, the estrogen can prevent glutamaterelated cell death by decreasing extracellular glutamate levels through an increased glutamate uptake capacity by astrocytes (Pawlak et al., 2005). The reported effects might be responsible for the ability of estrogen in reducing the detrimental action of ischemia/reperfusion on glutamate transporters thus limiting accumulation of extracellular glutamate and preventing the death of RGC. Acknowledgment Financial support from the Italian Ministry of Health (Rome) is acknowledged. References Adachi, K., Kashii, S., Masai, H., Ueda, M., Morizane, C., Kaneda, K., Kume, T., Akaike, A. and Honda, Y. (1998) Mechanism of the pathogenesis of glutamate neurotoxicity in
590 retinal ischemia. Graefes Arch. Clin. Exp. Ophthalmol., 236: 766–774. Amantea, D., Russo, R., Bagetta, G. and Corasaniti, M.T. (2005) From clinical evidence to molecular mechanisms underlying neuroprotection afforded by estrogens. Pharmacol. Res., 52: 119–132. Casson, R.J. (2006) Possible role of excitotoxicity in the pathogenesis of glaucoma. Clin. Exp. Ophthalmol., 34: 54–63. Clark, J.H. and Markaverich, B.M. (1983) The agonistic and antagonistic effects of short acting estrogens: a review. Pharmacol. Ther., 21: 429–453. Garcia-Segura, L.M., Azcoitia, I. and DonCarlos, L.L. (2001) Neuroprotection by estradiol. Prog. Neurobiol., 63: 29–60. Goodman, Y., Bruce, A.J., Cheng, B. and Mattson, M.P. (1996) Estrogens attenuate and corticosterone exacerbates excitotoxicity, oxidative injury, and amyloid beta-peptide toxicity in hippocampal neurons. J. Neurochem., 66: 1836–1844. Joo, C., Cho, K., Kim, H., Choi, J.S. and Oh, Y.J. (1999) Protective role for bcl-2 in experimentally induced cell death of bovine corneal endothelial cells. Ophthalmic Res., 31: 287–296. Kumar, D.M., Perez, E., Cai, Z.Y., Aoun, P., Brun-Zinkernagel, A.M., Covey, D.F., Simpkins, J.W. and Agarwal, N. (2005) Role of nonfeminizing estrogen analogues in neuroprotection of rat retinal ganglion cells against glutamate-induced cytotoxicity. Free Radic. Biol. Med., 38: 1152–1163. Lubahn, D.B., McCarty, K.S., Jr. and McCarty, K.S., Sr. (1985) Electrophoretic characterization of purified bovine, porcine, murine, rat, and human uterine estrogen receptors. J. Biol. Chem., 260: 2515–2526. Luo, X., Lambrou, G.N., Sahel, J.A. and Hicks, D. (2001) Hypoglycemia induces general neuronal death, whereas hypoxia and glutamate transport blockade lead to selective retinal ganglion cell death in vitro. Invest. Ophthalmol. Vis. Sci., 42: 2695–2705. Maggi, A., Ciana, P., Belcredito, S. and Vegeto, E. (2004) Estrogens in the nervous system: mechanisms and nonreproductive functions. Annu. Rev. Physiol., 66: 291–313. Muller, A., Maurin, L. and Bonne, C. (1998) Free radicals and glutamate uptake in the retina. Gen. Pharmacol., 30: 315–318. Nakazawa, T., Takahashi, H. and Shimura, M. (2006) Estrogen has a neuroprotective effect on axotomized RGCs through ERK signal transduction pathway. Brain Res., 1093: 141–149. Nucci, C., Tartaglione, R., Rombola`, L., Morrone, L.A., Fazzi, E. and Bagetta, G. (2005) Neurochemical evidence to implicate elevated glutamate in the mechanisms of high intraocular pressure (IOP)-induced retinal ganglion cell death in rat. Neurotoxicology, 26: 935–941. Osborne, N.N., Casson, R.J., Wood, J.P., Childlow, G., Graham, M. and Melena, J. (2004) Retinal ischemia: mechanisms of damage and potential therapeutic strategies. Prog. Retin. Eye Res., 23: 91–147.
Osborne, N.N., Ugarte, M., Chao, M., Childlow, G., Bae, J.H., Wood, J.P. and Nash, M.S. (1999) Neuroprotection in relation to retinal ischemia and relevance to glaucoma. Surv. Ophthalmol., 43(Suppl 1): s102–s128. Pawlak, J., Brito, V., Kuppers, E. and Beyer, C. (2005) Regulation of glutamate transporter GLAST and GLT-1 expression in astrocytes by estrogen. Brain Res. Mol. Brain Res., 138: 1–7. Quigley, H.A. (1996) Number of people with glaucoma worldwide. Br. J. Ophthalmol., 80: 389–393. Richards, D.A., Morrone, L.A., Bagetta, G. and Bowery, N.G. (2000) Effects of a-dendrotoxin and dendrotoxin k on extracellular excitatory amino acids and on electroencephalograph spectral power in the hippocampus of anaesthetised rats. Neurosci. Lett., 293: 183–186. Simpkins, J.W. and Dykens, J.A. (2008) Mitochondrial mechanisms of estrogen neuroprotection. Brain Res. Rev., 57: 421–430. Singer, C.A., Figueroa-Masot, X.A., Batchelor, R.H. and Dorsa, D.M. (1999) The mitogen-activated protein kinase pathway mediates estrogen neuroprotection after glutamate toxicity in primary cortical neurons. J. Neurosci., 19: 2455–2463. Singer, C.A., Rogers, K.L., Strickland, T.M. and Dorsa, D.M. (1996) Estrogen protects primary cortical neurons from glutamate toxicity. Neurosci. Lett., 212: 13–16. Tezel, G. (2006) Oxidative stress in glaucomatous neurodegeneration: mechanisms and consequences. Prog. Retin. Eye Res., 25: 490–513. Tezel, G., Yang, X. and Cai, J. (2005) Proteomic identification of oxidatively modified retinal proteins in a chronic pressureinduced rat model of glaucoma. Invest. Ophthalmol. Vis. Sci., 46: 3177–3187. Weaver, C.E., Jr., Park-Chung, M., Gibbs, T.T. and Farb, D.H. (1997) 17beta-Estradiol protects against NMDAinduced excitotoxicity by direct inhibition of NMDA receptors. Brain Res., 761: 338–341. Wise, P. (2003) Estradiol exerts neuroprotective actions against ischemic brain injury: insights derived from animal models. Endocrine, 21: 11–15. Yang, S.H., Liu, R., Wu, S.S. and Simpkins, J.W. (2003) The use of estrogens and related compounds in the treatment of damage from cerebral ischemia. Ann. N.Y. Acad. Sci., 1007: 101–107. Zaulyanov, L.L., Green, P.S. and Simpkins, J.W. (1999) Glutamate receptor requirement for neuronal death from anoxia–reoxygenation: an in vitro model for assessment of the neuroprotective effects of estrogens. Cell. Mol. Neurobiol., 19: 705–718. Zhou, X., Li, F., Ge, J., Sarkisian, S.R., Jr., Tomita, H., Zaharia, A., Chodosh, J. and Cao, W. (2007) Retinal ganglion cell protection by 17-beta-estradiol in a mouse model of inherited glaucoma. Dev. Neurobiol., 67: 603–616.
CHAPTER 40
17b-Estradiol prevents retinal ganglion cell loss induced by acute rise of intraocular pressure in rat Rossella Russo1, Federica Cavaliere1, Chizuko Watanabe2, Carlo Nucci3,4, Giacinto Bagetta1,5, Maria Tiziana Corasaniti4,6, Shinobu Sakurada2 and Luigi Antonio Morrone1,5, 1
Department of Pharmacobiology, University of Calabria, 87036 Arcavacata di Rende, Italy Department of Physiology and Anatomy, Tohoku Pharmaceutical University, Sendai, Japan 3 Physiopathological Optics, Department of Biopathology, University of Rome ‘‘Tor Vergata,’’ 00133 Rome, Italy 4 ‘‘Mondino-Tor Vergata’’ Center for Experimental Neurobiology, University of Rome ‘‘Tor Vergata,’’ 00133 Rome, Italy 5 Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University Center for Adaptive Disorders and Headache (UCADH), University of Calabria, 87036 Arcavacata di Rende, Italy 6 Department of Pharmacobiological Sciences, University ‘‘Magna Graecia’’ of Catanzaro, 88100 Catanzaro, Italy 2
Abstract: Glaucoma, is a progressive optic neuropathy often associated with increased intraocular pressure (IOP) and characterized by progressive death of retinal ganglion cells (RGCs). High acute rise of IOP is a model for retinal ischemia and may represent a model of acute angle closure glaucoma. Here we have used this experimental model in combination with a neurochemical and neuropathological approach to gain more insight in the neuroprotective profile of 17b-estradiol (E2), a steroid hormone, which has been shown to increase the viability, survival, and differentiation of primary neuronal cultures from different brain areas including amygdala, hypothalamus, and neocortex. Our data demonstrate that systemic administration of E2 significantly reduces RGC loss induced by high IOP in rat. In addition, pretreatment with E2, 30 min before ischemia, minimizes the elevation of glutamate observed during the reperfusion period. These effects seem to be in part mediated by the activation of the estrogen receptor, since a pretreatment with ICI 182-780, a specific estrogen receptor antagonist, partially counteracts the neuroprotection afforded by the estrogen. Keywords: glaucoma; excitotoxicity; oxidative stress; estrogens; microdialysis; neuroprotection
intraocular pressure (IOP) that is characterized by progressive death of retinal ganglion cells (RGCs) and consequent deterioration of the visual field. Several studies suggest that excitotoxicity occurs during retinal ischemia with subsequent loss of RGCs and that this process plays a role in the pathogenesis of ischemic retinopathy (Nucci et al., 2005; Casson, 2006). Glutamate functions as the major excitatory amino acid neurotransmitter
Introduction Glaucoma, one of the leading causes of blindness in the world (Quigley, 1996), is a progressive optic neuropathy often associated with increased
Corresponding author. Tel.: +39 0984 493054; Fax: +39 0984 493462; E-mail: [email protected]
DOI: 10.1016/S0079-6123(08)01144-8
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in the retina, but at high concentrations it becomes neurotoxic. The pivotal role of excessive glutamate in the mechanisms of retinal damage is also documented by the evidence that NMDA and non-NMDA receptor antagonists afford protection in experimental models of RGC death, both in vitro and in vivo (Adachi et al., 1998; Joo et al., 1999; Osborne et al., 1999; Nucci et al., 2005). Growing evidence also supports that oxidative stress is the leading mechanism of excitotoxic, glutamateinduced RGC loss in vitro (Luo et al., 2001) and in vivo (Nucci et al., 2005). Not only the interference with glutamate transmission may stem from the decrease of the retinal glutamate transport (Muller et al., 1998), but also glutamine synthetase, which converts glutamate to glutamine, is oxidatively modified in ocular hypertensive eyes (Tezel et al., 2005). In addition, retinal glutamate damage has been shown to be mediated in part through nitric oxide, a highly reactive radical species (Nucci et al., 2005). Based on previous observations, neuroprotection of RGC from excitotoxicity is becoming an important approach of glaucoma therapy. Interestingly, it has been recently demonstrated that 17b-estradiol (E2) minimizes RGC loss in DBA/2J mouse, an in vivo model of an inherited (pigmentary) glaucoma (Zhou et al., 2007), shows neuroprotective effect on axotomy-induced RGC death (Nakazawa et al., 2006), and protects RGC against glutamate cytotoxicity (Kumar et al., 2005). In addition, several studies showed that estrogens, a family of cholesterol-derived steroid hormones, are protective against various oxidative stress insults including excitotoxicity (Goodman et al., 1996; Singer et al., 1996, 1999; Weaver et al.,1997; Zaulyanov et al., 1999). Cumulative evidence from basic science and clinical research suggests that estrogens play a significant neuromodulatory and neuroprotective role in the brain, and this underlies their ability to ameliorate symptoms and decrease the risk of neurodegenerative conditions such as cerebrovascular stroke, Alzheimer’s disease, and Parkinson’s disease (see Amantea et al., 2005). The mechanisms underlying estrogen neuroprotection have not been completely elucidated and several mechanisms have been proposed to explain the neurotrophic and neuroprotective actions of estrogens, including modulation of synaptogenesis,
protection against apoptosis, anti-inflammatory activity, and increased cerebral blood flow (Garcia-Segura et al., 2001; Wise, 2003; Maggi et al., 2004). Using high IOP experimental model in rat, in combination with a neurochemical and neuropathological approach, we now report the neuroprotection afforded by systemic treatment with E2, showing that this hormone is able to prevent the glutamate-induced loss of RGC under these experimental conditions.
Methods Male, Wistar rats (250–300 g) (Charles River, Lecco, Italy) were maintained on a 12-h light–dark cycle. Before ischemia was induced, animals were anesthetized with chloral hydrate (400 mg kg–1, intraperitoneally (i.p.)). Corneal analgesia was achieved using topical drops of oxibuprocaine 0.4% (Novesina, Novartis Farma, Italy). Pupillary dilation was maintained using 0.5% tropicamide (Visumidriatic 0.5%, Visufarma, Italy). The anterior chamber of the right eye was cannulated with a 27-gauge infusion needle connected to a 500 mL plastic container of sterile saline, the IOP was raised to 120 mmHg for 50 min by elevating the saline reservoir. Retinal ischemia was confirmed by observing whitening of the iris and loss of the red reflex of the retina. Sham procedure was performed without the elevation of the bottle in control rats. Morphometric analysis Rats receiving ischemic insult or sham procedure were anaesthetized (chloral hydrate 400 mg kg–1, i.p.) and perfused through the left ventricle of the heart with 50 mL of heparinized phosphate buffered (pH 7.4) saline followed by 50 mL of 4% paraformaldehyde in phosphate buffered saline at 6 h, 12 h, 24 h, 48 h, and 7 days after reperfusion (n ¼ 6 per group). Two hours after the perfusion procedure had been completed, the eyes were enucleated and post-fixed in 4% paraformaldehyde for 72 h. Serial coronal sections, cut along the vertical meridian of the eye passing through the optic nerve head, were stained with hematoxylin
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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 2 mL 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 2 h 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
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 kg1, given i.p. twice daily), whereas test group received E2 (i.p., 0.2 mg kg1; n ¼ 3) 30 min before ischemia or the estrogen receptor antagonist, ICI 182-780 (i.p., 0.2 and 2 mg kg1; 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 kg1; n ¼ 5) and the 17a-isomer of estradiol (E2a, i.p., 0.2 mg kg1; n ¼ 3) 30 min before ischemia. ICI 182-780 (i.p., 0.2 and 2 mg kg1; 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 kg1), 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 kg1 (Table 1, Fig. 1), whereas it partially counteracted (15.18%) the effect of E2 at a dose of 2 mg kg1 (Table 1).
586 Table 1. Retinal ganglion cell (RGC) loss induced by acute high intraocular pressure is prevented by systemic treatment with 17bestradiol Experimental conditions
Number of RGC
Percentage vs. control
Control (sham-operated) Ischemia 17b-Estradiol (0.2 mg kg1)+ischemia ICI 182-780 (0.2 mg kg1)+17b-estradiol+ischemia ICI 182-780 (2 mg kg1)+17b-estradiol+ischemia
35.4370.08 25.5070.29# 32.9070.82 33.0870.32,y 30.0570.22
28.03 7.14 6.63 15.18
Elevated IOP-induced ischemia for 50 min was followed by a 24-h reperfusion period. Control animals (n ¼ 6) received injections of saline (1 mg kg1, given i.p. twice daily), whereas test group received i.p. 17b-estradiol (E2, n ¼ 3) 30 min before ischemia or the estrogen receptor antagonist, ICI 182-780 (i.p., n ¼ 3 per group), 1 h before injection of E2. Cell counting was performed in the ganglion cell layer of ischemic/reperfused and sham-operated rat retinas stained with hematoxylin and eosin. The number of RGC was counted in six areas of each section (n ¼ 6 for eye) using light microscopy. The data were expressed as mean7SEM per area, and were evaluated statistically for differences using the Student’s t-test. # p ¼ 0.000 vs. control. po0.01 vs. ischemia. y p ¼ 0.8 vs. E2+ischemia
Fig. 1. Retinal ischemia for 50 min followed by 24 h reperfusion reduces the number of cells in the retinal ganglion cell layer (B, n ¼ 6 rats) as compared to sham-operated rats (A, n ¼ 6). Systemic treatment with 17b-estradiol (C, i.p., 0.2 mg kg1, n ¼ 3 rats) prevents the tissue damage observed in (B). A pretreatment with ICI 182-780 (D, i.p., 0.2 mg kg1, n ¼ 3 rats), a specific estrogen receptor antagonist, failed to abrogate the neuroprotection afforded by 17b-estradiol. H&E staining. RGC: retinal ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer.
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 kg1 given i.p.; n ¼ 5 rats), 30 min before ischemia, did not
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Fig. 2. Neurochemical data obtained by intraocular microdialysis experiments in anesthetized rats (n ¼ 6) demonstrate that ischemia/ reperfusion insult increases intraretinal glutamate. The extracellular level of glutamate (GLU, solid line) from the retina tended to increase after the first 10 min of ischemia with a larger and statistically significant increase observed at 10 and 150 min after the reperfusion had started. Systemic administration of 17b-estradiol (i.p., 0.2 mg kg1, n ¼ 5 rats, dashed line), 30 min before ischemia, did not significantly affect the GLU peak increase observed at 10 min after ischemia, whereas it minimized the elevation of GLU observed during the reperfusion period. The baseline concentrations of GLU were the mean concentrations obtained by averaging the six samples collected consecutively at 10 min intervals immediately prior to the onset of ischemia and were used as basal values. Glutamate values (mM) are expressed as mean7SEM. Statistical significance was assessed by ANOVA followed by Dunnett’s test for multiple comparisons # and po0.05 vs. basal values; po0.001 vs. basal values.
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 kg1 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 kg1 (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 kg1, 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
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Fig. 3. Effect of 17b-estradiol (i.p., 0.2 mg kg1, n ¼ 5 rats) and of the estrogen receptor antagonist ICI 182-780 (0.2 mg kg1, n ¼ 5 rats) on levels of glutamate (GLU) observed at 10 and 150 min after reperfusion had started. Administration of 17b-estradiol (gray columns) significantly reduced the GLU increase observed after 10 and 150 min of reperfusion. ICI 182-780 (black columns) failed to counteract the effect on extracellular GLU levels afforded by 17b-estradiol. The white columns show the extracellular GLU levels obtained in ischemia/reperfusion (isch/rep) experiments (n ¼ 6). Data are expressed as mean7SEM percentage of basal values of GLU. The baseline concentrations of glutamate were the mean concentrations obtained by averaging the six samples collected consecutively at 10 min intervals immediately prior to the onset of ischemia. Data were tested for statistical significance with paired, two-tailed, Student’s t-test. po0.05 vs. isch/rep.
Fig. 4. Neurochemical data obtained by intraocular microdialysis experiments in anaesthetized rats (n ¼ 6) demonstrate that the extracellular level of glutamate (GLU, solid line) from the retina tended to increase after the first 10 min of ischemia with a larger and statistically significant increase observed at 10 and 150 min after the reperfusion had started. Systemic administration of 17a-estradiol (i.p., 0.2 mg kg1, n ¼ 3 rats, dashed line), 30 min before ischemia, did not significantly affect the GLU peak increase observed at 10 min after ischemia and after 10 min of reperfusion, whereas it minimized the elevation of GLU observed during the late reperfusion period. The baseline concentrations of GLU were the mean concentrations obtained by averaging the six samples collected consecutively at 10 min intervals immediately prior to the onset of ischemia and were used as basal values. Glutamate values (mM) are expressed as mean7SEM. Statistical significance was assessed by ANOVA followed by Dunnett’s test for multiple comparisons # and po0.05 vs. basal values; po0.001 vs. basal values.
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reperfusion, extracellular glutamate increases significantly in the retina of the ischemic eye and this is sensitive to the prevention afforded by systemic MK801 (Nucci et al., 2005). The present study shows that systemic pretreatment with E2, 30 min before ischemia, prevents glutamate increase during reperfusion and this is accompanied by minimization of RGC death at 24 h of reperfusion. Accordingly, recent data demonstrated that E2 minimizes RGC loss in DBA/2J mouse, an in vivo model of an inherited glaucoma (Zhou et al., 2007), has neuroprotective effect on axotomy-induced RGC death (Nakazawa et al., 2006), and protects RGC against glutamate cytotoxicity (Kumar et al., 2005). Here we monitored in vivo extracellular glutamate levels from rat retina during and after pressure-induced ischemia using an established microdialysis technique (Nucci et al., 2005). An increase of dialysate glutamate levels occurred during ischemia, followed by a more pronounced and statistically significant increase during the reperfusion phase. Interestingly, neurochemical data show that pretreatment with E2 fails to counteract glutamate increase typically observed during ischemia, while it significantly inhibits the subsequent increase observed during reperfusion. The mechanism underlying neuroprotection afforded by E2 is not known; however, under our experimental conditions, the effect of E2 on RGC seems to be mediated in part by the activation of the estrogen receptors. In fact, a pretreatment with high concentrations of a specific estrogen receptor antagonist, the compound ICI 182-780, partially counteracts the neuroprotection afforded by the estrogen. Interestingly, this high dose of ICI 182-780 is able to counteract partially the effect of E2 on extracellular levels of glutamate during reperfusion period, whereas a lower dose of ICI 182-780 fails to counteract any effect of E2 on extracellular glutamate levels during reperfusion. Particularly, the higher dose of ICI 182-780 prevents the effect of E2 in the early reperfusion phase, but not during the late phase. This experimental observation seems to suggest that in our experimental conditions, the increase of glutamate observed in the early phase of reperfusion is under the control of estrogen receptors whereas the accumulation of
glutamate in the late reperfusion phase seems to stem from an estrogen receptor-independent mechanism. Accordingly, we demonstrate that the E2a, a weak estrogen receptor agonist (Clark and Markaverich, 1983; Lubahn et al., 1985, Yang et al., 2003), does not affect the glutamate increase observed in the early phase of reperfusion but it is effective as E2 in minimizing the glutamate increase observed in the late reperfusion period. The latter result strengthens the hypothesis that this estrogenic effect is mediated via a mechanism that does not require binding to the cytosolic estrogen receptor. Interestingly, although many of the estrogen neuroprotective effects appear to be mediated through the activation of intracellular estrogen receptors (ER), ERa and ERb, several observations support the possibility that estrogens exert their potent neuroprotective effects through a mitochondrial mechanism (see Simpkins and Dykens, 2008). Increasing experimental evidence implicates failure of mitochondrial energy metabolism in the pathogenesis of neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases (see Tezel, 2006). Following a detrimental stimulus, estrogens preserve ATP production, prevent production of ROS, moderate excessive cellular and mitochondrial Ca2+ loading, and preserve mitochondrial membrane potential (see Simpkins and Dykens, 2008). Accordingly, the estrogen can prevent glutamaterelated cell death by decreasing extracellular glutamate levels through an increased glutamate uptake capacity by astrocytes (Pawlak et al., 2005). The reported effects might be responsible for the ability of estrogen in reducing the detrimental action of ischemia/reperfusion on glutamate transporters thus limiting accumulation of extracellular glutamate and preventing the death of RGC. Acknowledgment Financial support from the Italian Ministry of Health (Rome) is acknowledged. References Adachi, K., Kashii, S., Masai, H., Ueda, M., Morizane, C., Kaneda, K., Kume, T., Akaike, A. and Honda, Y. (1998) Mechanism of the pathogenesis of glutamate neurotoxicity in
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