Protection of neurons in the retinal ganglion cell layer against excitotoxicity by the N-acylethanolamine, N-linoleoylethanolamine

Abstract

Retinal ganglion cell (RGC) death is a hallmark of neurodegenerative diseases and disease processes of the eye, including glaucoma. The protection of RGCs has been an important strategy for combating glaucoma, but little clinical success has been reported to date. One pathophysiological consequence of glaucoma is excessive extracellular glutamate subsequently leading to excitotoxicity in the retina. Endocannabinoids, such as the N-acylethanolamine (NAE), arachidonylethanolamine (NAE 20:4), exhibit neuroprotective properties in some models of neurodegenerative disease. The majority of NAEs, however, are not cannabinoids, and their physiological function is not clear. Here, we determined whether the noncannabinoid NAE, linoleoylethanolamine (NAE18:2), protects neurons in the RGC layer against glutamate excitotoxicity in ex-vivo retina cultures. Using a terminal deoxynucleotidyl transferase-mediated dUTP (2'-deoxyuridine 5'-triphosphate) nick-end labeling (TUNEL) assay, we determined that NAE18:2 reduces the number of apoptotic RGC layer neurons in response to glutamate and conclude that NAE18:2 is a neuroprotective compound with potential for treating glaucomatous retinopathy.

Keywords: calcium signaling; eye; glaucoma; glutamate; immunocytochemistry; neuroprotection; vision.

Figure 1

Neurons in the RGC layer are sensitive to glutamate excitotoxicity. A) Exposure of retinas to 40 μM NAE 18:2 or ethanol vehicle does not affect viability of neurons in the RGC layer. Glutamate (100 μM) exposure, however, causes apoptotic cell death in the RGC layer of retina explants (red arrows). B) Quantitative summary data revealing the lack of toxicity of NAE 18:2 or its vehicle as well as the degree of cell death in the RGC layer measured as the number of TUNEL-positive cells in retinas exposed to glutamate. RGCs were identified by Thy 1.2 and neurofilament-68 kDa immunoreactivity and location., Notes: Scale bar: 50 μM. *denotes P < 0.05, as determined with a one-way analysis of variance test. Abbreviations: DIC, differential interference contrast; NAE, N-acylethanolamide; RGC, retinal ganglion cell; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP (2′-deoxyuridine 5′-triphosphate) nick-end labeling – green fluorescence.

Figure 2

NAE 18:2 protects RGC layer neurons from glutamate excitotoxicity. A) Exposure of retinas to 100 μM glutamate resulted in a dramatic increase in RGC layer neuron death (red arrows). Preincubation of retinas with NAE 18:2 for 6 hours prior to glutamate exposure resulted in a dose-dependent decrease in the number apoptotic, TUNEL-positive RGC layer neurons, with high physiological concentrations reducing the number of apoptotic neurons. RGCs were identified by Thy 1.2 immunoreactivity and location., B) Quantitative summary data for the NAE 18:2-mediated neuroprotection from glutamate toxicity as measured by TUNEL histochemistry. Notes: *denotes P < 0.05, as determined by a one-way analysis of variance test. Scale bar: 50 μM. Abbreviations: DIC, differential interference contrast; NAE, N-acylethanolamide; RGC, retinal ganglion cell; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP (2′-deoxyuridine 5′-triphosphate) nick-end labeling – green fluorescence.

Figure 3

NAE 18:2 preincubation is required for protection of RGC layer neurons against glutamate excitotoxicity. NAE 18:2 preincubation is critical for its neuroprotective effect. A) Retina explants were either pretreated for 24 hours (ON) with 40 μM NAE 18:2 prior to and during glutamate exposure or at the onset of glutamate exposure (no pretreatment). All glutamate exposures were for 24 hours, and NAE 18:2 was present during the glutamate exposure period. Note the lack of protection without pretreatment (red arrows). B) Quantitative summary data revealing the requirement for NAE 18:2 protection of RGC layer neurons against glutamate. Notes: *denotes P < 0.05, as determined by a one-way analysis of variance test. Scale bar: 50 μM. Abbreviations: DIC, differential interference contrast; NAE, N-acylethanolamide; ON, overnight; RGC, retinal ganglion cell; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP (2′-deoxyuridine 5′-triphosphate) nick-end labeling – green fluorescence.