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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