Caratterizzazione del profilo neurochimico, neuropatologico e neourofarmacologico di un modello sperimentale di glaucoma acuto

Abstract

Glaucoma is a leading cause of irreversibile blindness characterized by the progressive loss of retinal ganglion cells (RGCs). Elevated intraocular pressure is a major risk factor for glaucoma; however, medical therapies aimed to reduce intraocular pressure, are not always successful blocking the glaucoma‐associated retinal degeneration. This observation suggests that mechanisms other than intraocular pressure are also implicated in glaucomatous neurodegeneration. Experimental evidences suggest that alteration of glutamate homeostasis contributes to retinal dysfunction and neuropathy. However, the involvement of excitotoxicity in RGCs loss is still controversial. Therefore, the aim of the present study was to investigate the modulation of intravitreal glutamate levels in high intraocular pressure (IOP) induced ischemia, an established animal model that recapitulates features of acute angle closure glaucoma. Using this experimental model we monitored extracellular glutamate in the vitreous of rat before, during and after high IOPinduced ischemia. A significant increase of extracellular glutamate occurred during reperfusion, 10 and 150 minutes after ischemia. In our experimental setting, calpain activation is detected in the retina explants early after ischemia. Excess activation of calpain caused deleterious effects on RGCs, in fact we have observed 25.9% loss of cells in the RGCs layer at 7 days of the reperfusion. A pre‐treatment with coenzyme Q10 prevented retinal ganglion cells loss and inhibited the increase of glutamate observed in the vitreous during reperfusion. In addition, systemic pre‐treatment with 17α and 17β‐ estradiol, endowed with neuroprotective and antioxidant properties, minimized the elevation of glutamate observed during the reperfusion period. The reported effects seem to be only 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 effect afforded by the estrogen in the early phase of reperfusion. In order to investigate the mechanisms supporting the observed increase of extracellular glutamate levels we performed ex‐vivo experiments using synaptosomes prepared from the retina of rats subjected to high IOP‐induced ischemia. Continuous superfusion of synaptosomes allowed a direct measurement of release and mechanisms involved. These data obtained indicated a reduced involvement of glutamate transporters in the mechanisms of release induced by KCl 15 mM in ischemic synaptosomes. In addition, in the same experimental conditions also the uptake is reduced. This is supported by reduced expression of the neuronal trasporter GLT1. Altogether these data, straighten the role of glutamate and oxidative stress in ischemiainduced RGCs death and demonstrate that the observed increase of intravitreal glutamate is associated to a reduction of the uptake and GLT1 expression.