Apoptotic cell death in the mouse retinal ganglion cell layer is induced in vivo by the excitatory amino acid homocysteine

Homocysteine, an excitatory amino acid and a homolog of cysteine, induces n euronal cell death in brain via stimulation of N-methyl-D-aspartate (NMDA) receptors. It also selectively activates NMDA receptors of retinal ganglion cells, but it is not known if high levels of homocysteine are toxic to the se cells. The purpose of this study was to determine whether increased leve ls of homocysteine caused death of neurons in the ganglion cell layer: if s o whether this death occurred via an apoptotic mechanism and to determine t he consequences of simultaneous elevation of homocysteine and glutamate, a known retinal excitotoxin, on the viability of neurons of the ganglion cell layer. C57BL/6 mice were injected intravitreally with either homocysteine or glutamate/homocysteine combined (final concentrations: 25, 75, and 200 m uM): injection of glutamate (25 and 200 muM) served as a positive control. Eyes were harvested and cryosections prepared 5-6 days post-injection. Syst ematic morphometric analysis of retinas of mice injected with homocysteine indicated that the total number of cells in the ganglion cell layer decreas ed by about 23 % following exposure to 200 muM homocysteine. To determine w hether the neurons of the ganglion cell layer were dying by apoptosis, the TUNEL method was used and was confirmed by immunohistochemical studies of c aspase-3, known to be expressed at high levels during retinal ganglion cell apoptosis. Microscopic analysis revealed significantly more TUNEL-positive cells in the ganglion cell layer in homocysteine-injected eyes than in con tralateral PBS-injected eyes. Retinas injected with 75 and 200 muM homocyst eine displayed significantly more TUNEL-positive neurons in the ganglion ce ll layer (2 and 2.9, respectively) than PBS-injected retinas (0.25). In eye s injected simultaneously with homocysteine/glutamate, the number of apopto tic cells in the ganglion cell layer almost doubled that for homocysteine o r glutamate injections alone. Immunohistochemical analysis of activated cas pase-3 revealed numerous positively labelled neurons in the ganglion cell l ayer in homocysteine and homocysteine/glutamate-injected eyes, but not in P BS-injected eyes. Quantification of this data revealed a significantly grea ter number of caspase-3-positive neurons in the ganglion cell layer of reti nas injected with 75 and 200 muM homocysteine (2.9 and 4.4, respectively) t han for PBS-injected retinas (0.5). This confirms that death of neurons in the ganglion cell layer is occurring by apoptosis. The present study provid es the first evidence that homocysteine is toxic to neurons of the ganglion cell layer. In addition, it provides evidence that these retinal neurons a re dying by apoptosis and it demonstrates for the first time that excitotox ic damage to neurons of the ganglion cell layer is potentiated by simultane ous elevation of homocysteine and glutamate. These findings are relevant to retinal ganglion cell death characteristic of diabetic retinopathy, which is thought to be mediated by overstimulation of the NMDA receptor. (C) 2001 Academic Press.