TY - JOUR
T1 - Apoptotic cell death in the mouse retinal ganglion cell layer is induced in vivo by the excitatory amino acid homocysteine
AU - Moore, Pamela
AU - El-sherbeny, Amira
AU - Roon, Penny
AU - Schoenlein, Patricia V.
AU - Ganapathy, Vadivel
AU - Smith, Sylvia B.
N1 - Funding Information:
We thank Mrs Susan Johnson for her expert assistance in preparation of this manuscript. This work was supported by National Institutes of Health Grants EY13089, EY12830, and PA-93-48, Fight for Sight-Prevent Blindness America, an unrestricted award from Research to Prevent Blindness, Inc. to the Department of Ophthalmology, Medical College of Georgia and the Medical College of Georgia Research Institute, Georgia, GA, U.S.A.
PY - 2001
Y1 - 2001
N2 - Homocysteine, an excitatory amino acid and a homolog of cysteine, induces neuronal 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 these cells. The purpose of this study was to determine whether increased levels of homocysteine caused death of neurons in the ganglion cell layer; if so whether this death occurred via an apoptotic mechanism and to determine the 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 μM); injection of glutamate (25 and 200 μM) served as a positive control. Eyes were harvested and cryosections prepared 5-6 days post-injection. Systematic morphometric analysis of retinas of mice injected with homocysteine indicated that the total number of cells in the ganglion cell layer decreased by about 23% following exposure to 200 μM homocysteine. To determine whether the neurons of the ganglion cell layer were dying by apoptosis, the TUNEL method was used and was confirmed by immunohistochemical studies of caspase-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 contralateral PBS-injected eyes. Retinas injected with 75 and 200 μM homocysteine displayed significantly more TUNEL-positive neurons in the ganglion cell layer (2 and 2.9, respectively) than PBS-injected retinas (0.25). In eyes injected simultaneously with homocysteine/glutamate, the number of apoptotic cells in the ganglion cell layer almost doubled that for homocysteine or glutamate injections alone. Immunohistochemical analysis of activated caspase-3 revealed numerous positively labelled neurons in the ganglion cell layer in homocysteine and homocysteine/glutamate-injected eyes, but not in PBS-injected eyes. Quantification of this data revealed a significantly greater number of caspase-3-positive neurons in the ganglion cell layer of retinas injected with 75 and 200 μM homocysteine (2.9 and 4.4, respectively) than for PBS-injected retinas (0.5). This confirms that death of neurons in the ganglion cell layer is occurring by apoptosis. The present study provides the first evidence that homocysteine is toxic to neurons of the ganglion cell layer. In addition, it provides evidence that these retinal neurons are dying by apoptosis and it demonstrates for the first time that excitotoxic damage to neurons of the ganglion cell layer is potentiated by simultaneous 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.
AB - Homocysteine, an excitatory amino acid and a homolog of cysteine, induces neuronal 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 these cells. The purpose of this study was to determine whether increased levels of homocysteine caused death of neurons in the ganglion cell layer; if so whether this death occurred via an apoptotic mechanism and to determine the 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 μM); injection of glutamate (25 and 200 μM) served as a positive control. Eyes were harvested and cryosections prepared 5-6 days post-injection. Systematic morphometric analysis of retinas of mice injected with homocysteine indicated that the total number of cells in the ganglion cell layer decreased by about 23% following exposure to 200 μM homocysteine. To determine whether the neurons of the ganglion cell layer were dying by apoptosis, the TUNEL method was used and was confirmed by immunohistochemical studies of caspase-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 contralateral PBS-injected eyes. Retinas injected with 75 and 200 μM homocysteine displayed significantly more TUNEL-positive neurons in the ganglion cell layer (2 and 2.9, respectively) than PBS-injected retinas (0.25). In eyes injected simultaneously with homocysteine/glutamate, the number of apoptotic cells in the ganglion cell layer almost doubled that for homocysteine or glutamate injections alone. Immunohistochemical analysis of activated caspase-3 revealed numerous positively labelled neurons in the ganglion cell layer in homocysteine and homocysteine/glutamate-injected eyes, but not in PBS-injected eyes. Quantification of this data revealed a significantly greater number of caspase-3-positive neurons in the ganglion cell layer of retinas injected with 75 and 200 μM homocysteine (2.9 and 4.4, respectively) than for PBS-injected retinas (0.5). This confirms that death of neurons in the ganglion cell layer is occurring by apoptosis. The present study provides the first evidence that homocysteine is toxic to neurons of the ganglion cell layer. In addition, it provides evidence that these retinal neurons are dying by apoptosis and it demonstrates for the first time that excitotoxic damage to neurons of the ganglion cell layer is potentiated by simultaneous 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.
KW - Apoptosis
KW - Excitatory amino acids
KW - Glutamate
KW - Homocysteine
KW - Mouse
KW - Retinal ganglion cells
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U2 - 10.1006/exer.2001.1009
DO - 10.1006/exer.2001.1009
M3 - Article
C2 - 11428862
AN - SCOPUS:0034775253
SN - 0014-4835
VL - 73
SP - 45
EP - 57
JO - Experimental eye research
JF - Experimental eye research
IS - 1
ER -