Neuroprotective Effect of( ) -Tetrahydrocannabinol and Cannabidiol in N-Methyl-D-Aspartate-Induced Retinal Neurotoxicity Involvement of Peroxynitrite

In glaucoma, the increased release of glutamate is the major cause of retinal ganglion cell death. Cannabinoids have been demonstrated to protect neuron cultures from glutamate-induced death. In this study, we test the hypothesis that glutamate causes apoptosis of retinal neurons via the excessive formation of peroxynitrite, and that the neuroprotective effect of the psychotropic 9-tetrahydroxycannabinol (THC) or nonpsychotropic cannabidiol (CBD) is via the attenuation of this formation. Excitotoxicity of the retina was induced by intravitreal injection of N-methyl-Daspartate (NMDA) in rats, which also received 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (TEMPOL, a superoxide dismutase-mimetic), N-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), THC, or CBD. Retinal neuron loss was determined by TDT-mediated dUTP nick-end labeling assay, inner retinal thickness, and quantification of the mRNAs of ganglion cell markers. NMDA induced a doseand time-dependent accumulation of nitrite/ nitrate, lipid peroxidation, and nitrotyrosine (foot print of peroxynitrite), and a dose-dependent apoptosis and loss of inner retinal neurons. Treatment with L-NAME or TEMPOL protected retinal neurons and confirmed the involvement of peroxynitrite in retinal neurotoxicity. The neuroprotection by THC and CBD was because of attenuation of peroxynitrite. The effect of THC was in part mediated by the cannabinoid receptor CB1. These results suggest the potential use of CBD as a novel topical therapy for the treatment of glaucoma. (Am J Pathol 2003, 163:1997–2008) In glaucoma, reduction in intraocular pressure is frequently insufficient to prevent progression of visual field loss. Rather, glutamate-induced neurotoxicity plays an important role in glaucoma. Glutamate, an important neurotransmitter released from photoreceptor and bipolar cells, is present in high concentration in retinal ganglion cells. However, when excess glutamate is released into the surrounding medium because of compression or vascular occlusion, it is thought to activate a toxic response in adjacent cells. Excitotoxicity is mediated by overstimulation of the N-methyl-D-aspartate (NMDA) and non-NMDA receptors, a mechanism that has been demonstrated in the retina. Overstimulation of these receptors leads to excessive levels of intracellular calcium. This in turn leads to activation of nitric oxide synthase and excess accumulation of superoxides and nitric oxide (NO), causing lipid peroxidation, mitochondrial dysfunction, DNA damage, and the eventual cell death. Elevated glutamate levels have been found in the vitreous of glaucomatous patients and monkeys with glaucoma. Similarly, high glutamine, suggesting high extracellular glutamate, has been found in the retinal Müller cells of glaucomatous monkey eyes. The reaction product of superoxides and NO, peroxynitrite (ONOO ), was recently identified in the retinal ischemia-reperfusion injury. The causal role of ONOO in the glutamate-induced retinal excitotoxicity in glaucoma, however, has not been determined. In vitro and in vivo studies have demonstrated that ischemia-induced neurotoxicity can be reduced by antioxidants or by inhibitors of nitric oxide synthase. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl), a superoxide dismutase mimetic that permeates biological membranes and scavenges superoxides and hy-